DELIVERY OF THERMOSTABILIZED CHONDROTINASE ABC

ENHANCES AXONAL SPROUTING AND

FUNCTIONAL RECOVERY AFTER SPINAL CORD INJURY

A Dissertation
Presented to
The Academic Faculty

by

Hyun-Jung Lee

In Partial Fulfillment
of the requirements for the Degree
Doctor of Philosophy in the
School of Biomedical Engineering

Georgia Institute of Technology

October 2009

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 DELIVERY OF THERMOSTABILIZED CHONDROTINASE ABC

ENHANCES AXONAL SPROUTING AND

FUNCTIONAL RECOVERY AFTER SPINAL CORD INJURY

Dr. Ravi V. Bellamkonda, Advisor Dr. Robert J. McKeon

School of Biomedical Engineering Department of Cell Biology
Georgia Institute of Technology Emory University

Dr. Andrés J. García Dr. Niren Murthy

School of Mechanical Engineering School of Biomedical Engineering
Georgia Institute of Technology Georgia Institute of Technology

Dr. Andreas Bommarius

School of Chemical & Biomolecular Engineering
Georgia Institute of Technology

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 ACKNOWLEDGMENTS

First of all, I would like to acknowledge my advisor, Prof. Ravi V. Bellamkonda, for

his guidance over the years. He encouraged me to continue my research career, provided a

great environment, and always will be a good mentor. I feel lucky to have a chance to work

with him.

I also would like to acknowledge my thesis committee members, Prof. Andreas

Bommarius, Prof. Andrés J. García, Prof. Niren Murthy and Prof. Robert J. McKeon. I am

thankful for their guidance and insight for this study.

I was fortunate to meet my great labmates. I would like to express my appreciation to

all members of the Neurological Biomaterials and Cancer Therapeutics Laboratory. The

memories of the times we have shared will be in my mind forever and I will wish the best of

luck for all of you in your career and beyond.

I also like to express my appreciation to my sincere friends: Sungmoon, Moon and

Shannon who always emotionally supported me, listen to me and were there for me

whenever I needed them.

My parents and my brother always provide me unconditional love and any supports

and prayed for me every morning. I know they are always with me and in my mind wherever

I stay.

Without any of you, I could not get though this journey.

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 TABLE OF CONTENTS

ACKNOWLEDGMENTS ................................................................................................. iii

SUMMARY ..................................................................................................................... xvi

CHAPTER

INTRODUCTION………………………………………………………………………...1
1.1. STATEMENT OF PROBLEM ............................................................................ 1

1.2. HYPOTHESIS ..................................................................................................... 2

1.3. OBJECTIVES ...................................................................................................... 4

1.4. REFERENCES .................................................................................................... 5

RELEVANT BACKGROUND ...........................................................................................6

2.1 CELLULAR AND MOLECULAR RESPONSES AFTER SCI ......................... 7

2.2 CSPG-MEDIATED INHIBITION AFTER SCI AND CURRENT

TREATMENTS ................................................................................................... 8

2.2.1 CHONDROTIN SULFATE PROTEOGLYCANS ................................. 9

2.2.2 CHONDROITINASE ABC ................................................................... 10

2.2.3 DELIVERY OF CHONDROITIANSE ABC FOR AXONAL

REGENERATION AND LIMITATIONS ............................................ 15

2.3 THERAPEUTIC STRATEGIES TO IMPROVE THERMAL STABILITY OF

PROTEINS ........................................................................................................ 16

2.3.1 PROTEIN ENGINEERING................................................................... 17

2.3.2 USAGE OF COSOLVENTS IN AQUEOUS SYSTEM ....................... 18

2.3.2.1 TREHALOSE AS A PROTEIN STABILIZER .........................19

2.3.2.2 THE POSSIBLE MECHANISMS OF TREHALOSE

STABILIZATION OF CHABC…………………….…………20

2.3.3 PROTEIN STABILIZATION BY PEGYLATION……………………...22

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 2.4 THERAPEUTIC STRATEGIES FOR CONTROLLED RELEASE OF
AGENTS BY DELIVERY CARRIERS ............................................................ 26

2.4.1 POLYMER NANOPARTICLE ............................................................. 27

2.4.2 LIPID MICROTUBES........................................................................... 27

2.5 PHARMACOLOGICAL STRATEGIES FOR SCI……………………...……29

2.5.1 NEUROPROTECTIVE THERAPY TO TREAT ACUTE SCI……….29

2.5.2 NERVE GROWTH FACTORS: NEW POSSIBLE THERAPEUTIC

STRATEGY……………………………………………………………31

2.6 CONCLUSIONS................................................................................................ 32

2.7 REFERENCES .................................................................................................. 34

IMPROVEMENT OF THERMOSTABILITY OF CHONDROITINASE ABC

ENZYMATIC ACTIVITY AND DEVELOPMENT OF LIPID
MICROTUBE AND HYDROGEL MEDIATED DELIVERY
SYSTEM ..........................................................................................................45

3.1 INTRODUCTION ............................................................................................. 46

3.2 MATERIALS AND METHODS ....................................................................... 48

3.2.1 ENZYMATIC ACTIVITY ASSAY WITH SDS-PAGE ...................... 48

3.2.2 SILVER STAINING PROCEDURE ..................................................... 50

3.2.3 ENZYMATIC ACTIVITY ASSAY WITH DMMB ............................. 50

3.2.4 FABRICATION OF LIPID MICROTUBES......................................... 51

3.2.5 PREPARATION OF AGENT-LOADED LIPID MICROTUBES........ 52

3.2.6 ENZYAMTIC ACTIVITY ASSAY OF POST-RELEASED

CHONDROTINASE ABC FROM LIPID
MICROTUBES/HYDROGEL-MICROTUBE DELIVERY SCAFFOLD
................................................................................................................ 53

3.2.7 CHABC RELEASE PROFILES FROM LIPID MICROTUBES….….54

3.2.8 NT-3 RELEASE PROFILES FROM LIPID MICROTUBES .............. 55

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 3.2.9 ANALYSIS OF TEMPERATURE DEPENDENT CONFORMATION
OF CHABC ............................................................................................ 56

3.3 RESULTS .................................................................................................... 56

3.3.1 FUNCTIONAL STABILITY OF CHABC AT BODY TEMPERATURE

(37 ºC) WITHOUT THERMAL STABILIZATION ............................. 56

3.3.2 TREHALOSE SIGNIFICANTLY ENHANCES THERMOSTABILITY

OF CHABC ENZYMATIC ACTIVITY ............................................... 57

3.3.3 TEMPERATURE STABILIZATION OF CHABC BY TREHALOSE IS

DUE TO CONFORMATIONAL STABILITY ..................................... 59

3.3.4 LIPID MICROTUBE ENCAPSULATED CHABC IS

BIOLOGICALLY ACTIVE FOR 2 WEEKS........................................ 63

3.3.5 RELEASE PROFILE OF MICROTUBE ENCAPSULATED NT-3

CONFIRMS A SUSTAINED DELIVERY FOR 2 WEEK……………65

3.4 DISCUSSION .................................................................................................... 65

3.5 CONCLUSIONS................................................................................................ 69

3.6 REFERENCES .................................................................................................. 70

DELIVERY OF THERMOSTABILIZED CHABC BY IMPLANTING

HYDROGEL-MICROTUBE SCAFFOLDS AFTER SPINAL CORD
INJURY AND EXAMINATION OF CELLULAR AND
MOLECULAR RESPONSES: SHORT TERM STUDY ................................74

4.1 INTRODUCTION ............................................................................................. 75

4.2 MATERIALS AND METHODS ....................................................................... 78

4.2.1 FABRICATION OF MICROTUBE-HYDROGEL SCAFFOLDS ....... 78

4.2.2 TOPICAL DELIVERY OF HYDROGEL-MICROTUBE SCAFFOLDS

IN A DORSAL OVER HEMISECTION INJURY MODEL ................ 78

4.2.3 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY OF

SPINAL CORDS ................................................................................... 82

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 4.2.4 QUANTITATIVE ANALYSIS OF CSPG DIGESTION AND
ASTROCYTE RESPONSE ................................................................... 84

4.2.5 STATISTICAL ANALYSIS ................................................................. 86

4.3 RESULTS .......................................................................................................... 86

4.3.1 SUSTAINED DELIVERY OF ENCAPSULATED CHABC DIGESTS

CSPGS EFFECTIVELY IN VIVO ......................................................... 86

4.3.2 QUANTIFICATION OF 3B3, CS-56 AND GFAP

IMMUNOSTAINING....……………………………………………….89

4.4 DISCUSSION .................................................................................................... 92

4.5 CONCLUSIONS................................................................................................ 95

4.6 REFERENCES .................................................................................................. 96

DELIVERY OF THERMOSTABILIZED CHABC AND NT-3 AND

EVALUATION OF AXONAL REGENERATION AND
FUNCTIONAL RECOVERY AFTER SPINAL CORD INJURY:
LONG TERM STUDY ....................................................................................99

5.1 INTRODUCTION ........................................................................................... 100

5.2 MATERIALS AND METHODS ..................................................................... 103

5.2.1 TOPICAL DELIVERY OF HYDROGEL-MICROTUBE SCAFFOLDS

IN A DORSAL OVER HEMISECTION MODEL ............................. 103

5.2.2 RETROGRADE NEUROAL TRACER INJECTION INTO THE

SCIATIC NERVE ................................................................................ 104

5.2.3 BEHAVIORAL ANALYSIS ............................................................... 105

5.2.4 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY ..... 107

5.2.5 QUANTITATIVE ANALYSIS OF AXONAL SPROUTING............ 108

5.2.6 STATISTICAL ANALYSIS ............................................................... 109

5.3 RESULTS ........................................................................................................ 109

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 5.3.1 LEVELS OF CSPG DEPOSITION AFTER SUSTAINED DELIVERY
OF CHABC AT 6 WEEKS.................................................................. 109

5.3.2 SUSTAINED DELIVERY OF CHABC AND NT-3 IMPROVES

LOCOMOTOR FUNCTION ............................................................... 110

5.3.3 SUSTAINED DELIVERY OF CHABC AND NT-3 PROMOTES

SPROUTING ....................................................................................... 115

5.3.3.1 AXONAL SPROUTING AROUND THE LESION AREA:

CTB-LABELED FIBER ...........................................................115

5.3.3.2 AXONAL SPROUTING AROUND THE LESION AREA: 5-

HT- IMMUNOREACTIVE FIBERS ......................................118

5.4 DISCUSSION .................................................................................................. 121

5.5 CONCLUSIONS.............................................................................................. 126

5.6 REFERENCES ................................................................................................ 128

CONCLUSION AND FUTURE PERSPECTIVES ........................................................131

6.1 OPTIMIZATION OF CHABC TREATMENT FOR CLINICAL

APPLICATION ............................................................................................... 131

6.2 EFFECTS OF CHABC ON NERVE TISSUE AND IMMUNE SYSTEM .... 132

6.3 LONGER MICROTUBE: OPTIMIZATION OF DELIVERY VEHICLE ..... 133

6.4 OPTIMIZATION OF DOSAGE...................................................................... 134

6.5 LONGER IN VIVO STUDY ............................................................................ 135

6.6 RATE AND AMOUNT OF CSPG DEPOSITION IN VIVO AFTER SCI ..... 136

6.7 COMBINATION STRATEGIES: CHABC AND STEM CELL

TRANSPLANTATION ................................................................................... 136

6.8 DIFFERENCES BETWEEN HUMAN CASES AND RAT INJURY MODEL

………...…………………………………………………………………....…138

6.9 RESERENCES……………………………………….…………….………...141

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 LIST OF TABLES

Table 1. Experimental design of 2 week study with notation ...........................................82

Table 2. Experimental design of 45 days study with notation .........................................105

Table 3. A comparison between human and rat spinal cord. ...........................................139

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 LIST OF FIGURES

Figure 2.1. Structure of chondroitin sulfate proteoglycan (CSPG). (A) versican, a

large CSPG belonging to the lectican family and (B) decorin, a small
CSPG of extracellular matrix (Figure modified from
http://web.virginia.edu/Heidi/chapter9/chp9.htm). ..........................................11

Figure 2.2. Schematic structure of various CSPGs and hyaluronan. Figure from
Gilbert et al., 2005. ..........................................................................................12

Figure 2.3. Ribbon drawing of chondroitinase ABC I (figure from (Huang et al.,
2003)); the N-terminal (green), middle catalytic (blue) and C-terminal
domain (yellow), from top to bottom...............................................................14

Figure 2.4. Structure of trehalose. ......................................................................................20

Figure 2.5 Ploy(ethylene glycol).......................................................................................23

Figure 2.6 Schematic of PEGylated protein. The circles represent the water cloud
recruited by the ether-oxygen groups of the PEG polymer. Figure
from Veronese and Mero, 2008. ......................................................................24

Figure 3.1. Micrograph of lipid microtubes formed from DC8,9PC lipid in bright
field microscopy, scale bar is 50 µm. Figure from Meilander et al.,
2001..................................................................................................................52

Figure 3.2. Histogram of microtube length distribution. The average length is

about 37 µm, total number of microtubes from 1 mg of lipid is
approximately 1 × 108 and total inside volume of microtubes from 1
mg of lipid is 0.75 µl. .......................................................................................53

Figure 3.3. SDS-PAGE assay for chABC enzymatic activity after 1 day pre-
incubation and 1 week of pre-incubation at 37 ºC with and without
trehalose. Lane 1: fresh chABC + decorin, Lane 2: 1 day pre-incubated
chABC + decorin, Lane 3: intact decorin, Lane 4: chABC, Lane 5: 1
week pre-incubated chABC + decorin, and Lane 6: 1 week pre-
incubated chABC with trehalose + decorin. Thermostability of chABC
was enhanced with trehalose and trehalose-chABC still retained the
ability to digest decorin after 1 week incubation at body temperature. ...........57

x
Figure 3.4. chABC enzymatic activity assay with different concentrations of
trehalose by SDS-PAGE. Each lane represents 1) 20 mM, 2) 50 mM,
3) 100 mM, 4) 250 mM, 5) 500 mM and 6) 1M of trehalose-chABC
with decorin after 2 weeks of pre-incubation at 37 ºC.....................................58

Figure 3.5. chABC enzymatic activity test after 4 weeks of pre-incubation with
and without trehalose at 37 ºC by SDS-PAGE. Lane 1: 4 weeks pre-
incubated chABC with trehalose + decorin, Lane 2: 4 weeks pre-
incubated chABC + decorin, Lane 3: fresh chABC + decorin, Lane 4:
decorin, Lane 5: fresh chABC + decorin, Lane 6: fresh P‟ase +
decorin, and Lane 7: fresh P‟ase (out of molecular weight range from
gel; ~28 kDa). The trehalose-thermostabilized chABC still retained its
activity to degrade CS-GAG of decorin after 4 weeks of pre-incubation
at body temperature and P‟ase had no effect on decorin CS-GAG. ................58

Figure 3.6. Kinetic analysis of chABC deactivation by DMMB assay. The X axis
represents days and the Y axis represents percentage of digested
decorin. Asterisks denote a significant difference from chABC in 1X
PBS (P < 0.05) and data represent mean ± SEM. The dotted line
represents the calculated deactivation curve of chABC in 1x PBS. Data
are mean ± SEM. chABC in 1X PBS (×) loses most activity within 5
days, and in contrast, chABC in 1 M trehalose (Δ) retains its activity to
degrade decorin CS-GAG up to 15 days..........................................................61

Figure 3.7. The normalized thermal denaturation curves (fraction unfolded) of

chABC in pH 7.4 were measured by the changes of the absorbance at
222nm. The dashed line represents chABC in 50 mM sodium
phosphate buffer and the solid line represents chABC in 1M trehalose
solution. Tm of chABC in 1 M trehalose solution was 64.2 °C and the
Tm of chABC dissolved in buffer solution was 56.2 °C. Therefore, the
increment in the midpoint of transition, ΔTm, between the two
conditions is 8 °C and can be attributed to the presence of trehalose. .............62

Figure 3.8. Enzymatic activity of post-released chABC from lipid microtubes. (A)
Quantification of the amount of CS-GAGs that remained after
digestion of decorin with the released enzymes by DMMB assay.
chABC (Δ) and P‟ase (×) with trehalose/microtubes. The Y-axis
represents percentage of digested decorin and the X-axis represents
time. All data points are significantly different (P < 0.05; mean ±

xi
 SEM). (B) SDS-PAGE assay of chABC released from
trehalose/microtube + decorin..........................................................................64

Figure 3.9. Release profile of NT-3 from the hydrogel-microtube delivery

scaffold ………………………………………………………………………65

Figure 4.1. Schematic of spinal cord injury model and delivery of enzyme to
lesion site. The 1 % SeaPrep agarose gel-microtube-scaffold is
implanted on top of the lesion and covered with stiffer 0.7% SeaKem
agarose gel to keep the scaffold in place. ........................................................80

Figure 4.2. Schematic of gel cooling system. Tubing runs from the nitrogen gas
tank to a Styrofoam box containing 100% ethanol and dry ice, and
runs through an aluminum rod inside a tube containing dry ice to keep
the nitrogen gas cool. The cooled nitrogen gas was applied over the
stiffer agarose gel solution for in situ gelation, which covers the top of
the gel-microtube delivery scaffold. Figure from Jain et al., 2006. .................80

Figure 4.3. This figure demonstrates the topical delivery model applied into the
spinal cord lesion site. A) Exposed intact spinal cord at T10 level after
a single laminectomy. B) Dorsal over hemisection injury made with a
single cut to the spinal cord column. C) Gel scaffold, embedded with
chABC loaded microtubes, implanted on top of the lesion site. D) The
implanted hydrogel-microtube delivery scaffold was covered with
stiffer agarose gel to stabilize its location on top of the lesion site. (R =
rostral, C = caudal) ...........................................................................................81

Figure 4.4. Micrographs of the GFAP immunostained tissue and method of image
analysis with a custom developed MATLAB program. A) 4x
immunostained image. The solid line represents the lesion boundary
defined by GFAP immunoreactivity and the boxed areas denote
regions selected for quantification. Scale bar is 500 µm. B) One of the
boxes in figure A is expanded at 20x magnification to show analysis of
fluorescent intensity using line profiles. The X axis represents distance
from the lesion interface in pixel unit. The lines were generated from
the lesion interface into the tissue, averaged and displayed as a
function of distance from the interface to between experimental
groups. Scale bar is 100 µm. ............................................................................85

Figure 4.5. Immunohistological analysis of CSPG digestion in vivo (A-F). Images

were taken right next to the lesion boundary of no treatment (A, B and

xii
 C) and MTC treatment (hydrogel-microtube delivery scaffold loaded
with chABC/ 1M trehalose; D, E, and F) animals. CS-56-IR for intact
CSPGs (A and D). 3B3-IR for digested CSPGs (B and E). WFA
staining for perineuronal nets (C and F). The intensity of CS-56-IR and
WFA is inversely proportional to 3B3-IR. Arrows in (C) indicate
WFA-PNNs. Scale bar is 100µm. ....................................................................88

Figure 4.6. Quantitative image analysis of 3B3-IR and CS-56-IR fluorescent

intensity. The X axis represents each experimental condition treated
for animal groups. The Y axis represents the relative fluorescent
intensity of IR. The relative fluorescent intensity was measured alone
the lesion boundary, and mean value was obtained and averaged for
each animal. (A) 3B3-IR quantitative analysis. Asterisk denotes
significant increase of 3B3-IR in MTC treatment compared to all other
treatments (p<0.05). (B) CS-56-IR quantitative analysis. Asterisks
denote significant decrease of CS-56-IR in MTC treatment when
compared to NoT and STP treatments (p<0.05). No significant
differences were observed among other treatments. The data represent
the mean ± SEM...............................................................................................90

Figure 4.7. Quantitative image analysis of GFAP-IR fluorescent intensity by line

profile. The X axis represents distance from the lesion interface into
the spinal cord in µm and 0 represents the lesion interface delineate the
border of the astroglial scar lining the lesion site. The Y axis
represents the relative fluorescent intensity of GFAP-IR. Overall the
intensity of GFAP-IR decreased from the lesion interface into the cord
as a function of distance. The GFAP-IR was lower in the MTC treated
group than other control groups. The IR intensity was analyzed by
dividing into three bins, 0-100, 100-300 and 300-500. Asterisks denote
a significant difference between the MTC treated group and other
control groups; NoT, STP and MT in 0-100 µm and 100-300 µm (P
<0.05). The data represent the mean. ...............................................................91

Figure 5.1. CatWalk raw data in false color mode. White boxes represent
abnormal hindpaw print patterns. The dotted lines represent the stride
length defined as a distance between consecutive steps with the same
limb and the solid line represents the base of support defined as a
distance between the two hind paws. (dark red – left hindpaw, light red

xiii
 – left forepaw, dark green – right hindpaw and light green – right
forepaw) .........................................................................................................106

Figure 5.2. Micrograph of CS-56-IR around the lesion site at 6 weeks. (A) MTCN
and (B) MTP. CSPG-IR is significantly less in the MTCN treated
animals compared to MTP treated controls. Scale bar is 100 µm. ................110

Figure 5.3. CatWalk raw data in false color mode, (A) Sham, (B) MTCN and (C)
MTP treated animals on the walkway at 6 weeks. All animal groups
show a normal step sequence. The white box in the figure C represents
an abnormal hindpaw print. Red represents left paw prints and green
represents right paw prints (dark red – left hindpaw, light red – left
forepaw, dark green – right hindpaw and light green – right forepaw). ........113

Figure 5.4. Stride length analysis for locomotion functional recovery. Data are
mean ± SEM and asterisk denotes statistical significance between
MTCN and MTP, and between MTCN and GC (P <0.05). Sham
showed significant differences († < 0.05) compared to all other
conditions throughout the testing period, except for MTCN at 4 and 6
weeks..............................................................................................................114

Figure 5.5. Micrographs of CTB labeled fibers (green) at the lesion site at 6
weeks. The dashed lines represent the lesion interface. (A) MTP and
(B) MTCN at 10 x magnification. Scale bar is 500 µm (C) An
expanded figure from the white box 0 to 0.5 mm interval in figure A,
and (D) an expanded figure from the white box in 0.5 to 1 mm interval
figure B at 20 x magnification. Arrows represent CTB labeled fibers.
Scale bar is 100 µm. .......................................................................................116

Figure 5.6. Quantification of CTB+ axon growth. The Y axis represents the
percentage of crossed axons at the distance to the lesion interface and
the X axis represents distance to the lesion (mm). The data represent
the mean ± SEM. Asterisks denote a significant difference compared
with MTCN (P<0.05). ....................................................................................117

Figure 5.7. Immunohistological analysis of 5-HT-IR fibers. (A) Micrograph of 5-

HT at 4× magnification in the MTCN treated animal tissue. The boxed
areas in A denote regions selected for quantification and the solid
white line represents the lesion interface. More fibers were located
rostral versus caudal to the lesion. Scale bar is 500 µm. (B, C)

xiv
 Expanded figures from the white box in figure A. Serotonergic
innervations rostral to the lesion in MTP (B) and MTCN (C) animals
at 20× magnification. MTCN treated animal has higher fluorescent
intensity and also had extended closer to the lesion site than MTP
treated animal. Scare bar is 100 µm. ..............................................................119

Figure 5.8. Quantitative analysis of 5-HT-IR intensity for the stained spinal cord.
Quantification demonstrated that caudal to the lesion, MTCN showed
significantly (* p <0.05) increased 5-HT-IR compared to all other
treatments, and rostral to the lesion, MTCN showed significantly (* p
<0.05) increased 5-HT-IR compared to all other treatments except for
MTC. Data are mean ± SEM. ........................................................................120

Figure 6.1. Histogram of microtube length distribution with a modified fabrication

procedure. The average length is about 100 µm. ...........................................134

xv
 SUMMARY

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth

inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury

(SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion

of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising

results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal

regeneration and promote functional recovery when delivered at the site of injury. However,

chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity

rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions

with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity.

Maintaining these infusion systems is invasive and clinically problematic.

In this dissertation, three studies are reported that demonstrate our strategy to

overcome current limitations of using chABC and develop a delivery system for facilitating

chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding

trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo.

Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational

change of the enzyme was measured via circular dichroism (CD) with and without trehalose.

When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4

weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a

sustained release and showed that chABC released from the delivery system is still

functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-

xvi
microtube system was used to locally deliver chABC over two weeks at the lesion site

following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and

chABC loaded lipid microtubes was implanted at the top of the lesion site immediately

following injury. To determine effectiveness of topical delivery of thermostabilized chABC,

animal groups treated with single injection or gel scaffold implantation of chABC and

penicillinase (P‟ase) were included as controls. Two weeks after surgery, the functionality of

released chABC and the cellular responses were examined by immunohistological analysis

with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results

demonstrated that thermostabilized chABC was successfully delivered slowly and locally

without the need for an indwelling catheter by using the hydrogel-microtube delivery system

in vivo. The results demonstrated that released chABC from the gel scaffold effectively

digested CSPGs, and therefore, there were significant differences in CSPG digestion at the

lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and

controls. Third, a long term in vivo study (45 days) was conducted to examine axonal

sprouting/regeneration and functional recovery with both a single treatment each of

microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the

hydrogel-microtube delivery system. Over the long term study period, the treated animals

showed significant improvement in locomotor function and more sprouting of cholera toxin

B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers

around the lesion site.

We demonstrated that this significant improvement of chABC thermostability

facilitates the development of a minimally invasive method for sustained, local delivery of

chABC that is potentially a useful and effective approach for treating SCI. In addition to that,

xvii
we demonstrated that combinatorial therapy with chABC and neurotrophic factors could

provide a synergistic effect on axonal regrowth and functional recovery after SCI.

xviii
 CHAPTER 1

INTRODUCTION

1.1. STATEMENT OF PROBLEM

Unlike in the peripheral nervous system (PNS) or embryonic nervous system, in the

central nervous system (CNS) the severed axons fail to regrow through the lesion site, even

though the spared neurons survive for years.

There are approximately 255,000 people living with SCI in the United States and

there are 12,000 new cases added each year. After injury to the central nervous system, the

lesioned axons fail to re-grow and recover function (Schwab and Bartholdi, 1996). The

extent of sensory and motor function loss after spinal cord injury (SCI) varies depending on

the level of injury, and often results in permanent functional loss. Complete neurological

recovery after SCI is experienced by less than 1% of patients. Since 2000, the most frequent

category of spinal cord injury is incomplete tetraplegia (34.1%), followed by complete

paraplegia (23.0%), complete tetraplegia (18.3%), and incomplete paraplegia (18.5%). The

cost for treatment is up to $ 0.7 million per patient in the first year, and the lifetime cost is up

to $ 3 million (National Spinal Cord Injury Statistical Center, 2008). The cellular and

molecular mechanisms of the inflammation process, cell death, the mechanism of axon

growth failure and receptor/targeting relationships are all active areas of research. While

many groups are investigating a number of strategies to encourage axonal regeneration, a

breakthrough clinical therapy has yet to be developed.

1
 A challenge in the environment of the injured spinal cord is that the inhibitory

molecules create a non-permissive environment for axonal regrowth and it results in a failure

of axonal regeneration and complete functional recovery. chABC, which digests CS-GAGs

on CSPGs, has shown promise as a therapeutic agent for SCI treatment (Yick et al., 2000;

Krekoski et al., 2001; Bradbury et al., 2002) and CNS regeneration (Moon et al., 2001; Fox

and Caterson, 2002; Pizzorusso et al., 2002). However there are crucial limitations and

difficulties to be applied for clinical treatment; chABC is thermally very unstable and it loses

its enzymatic activity quickly at body temperature. Therefore, multiple injection of chABC or

mini-pump/catheter-mediated delivery system has been used to provide fresh chABC in vivo

for long periods. However, these infusion systems are invasive and require much effort to

maintain. In this study, an alternative strategy for chABC delivery in vivo is developed and

tested alone or in combination with neurtophin-3 (NT-3) to enhance axonal regeneration after

SCI.

1.2. HYPOTHESIS

The central hypothesis of this dissertation is that the digestion of CSPG by

chondroitinase ABC will promote axonal regeneration and functional recovery after SCI. The

chABC digestion will remove the inhibitory effect of CSPGs limiting axonal regeneration

after CNS injury and provide a permissive substrate for axonal outgrowth. We hypothesized

that delivery of thermostabilized chABC using our slow and local delivery system will

provide a sufficient amount of bioactive chABC to effectively digest CSPGs at the lesion site.

Also, the delivery method will be more efficient than single injection of an equal amount of

chABC to digest CSPGs, and it will enhance axonal sprouting/regeneration and induce

2
behavioral recovery. Therefore, our slow local delivery system could be an alternative

method to current mini-pump/catheter delivery methods, which are invasive and infection-

prone because they are chronically implanted. We believe that a combination strategy of

chABC and neurtophin-3 using the slow release delivery system will encourage even more

axonal regeneration and functional recovery.

It is important to achieve following design criteria to demonstrate our hypothesis: 1)

thermostabilized chABC; 2) a delivery vehicle for slow release in a temporally controlled

manner; and 3) a non-pump/single administration method in a spatially controlled manner.

Strategies to solve these challenges are: 1) protein stabilizers; 2) lipid microtube and

hydrogel mediated delivery system; and 3) a topical delivery model to the dorsal over

hemisection injury.

3
1.3. OBJECTIVES

The overall purpose of the work described in this thesis is to develop an approach to

successfully use chABC as a therapeutic agent. To overcome limitations of chABC treatment

for SCI, it is necessary to improve thermostability of chABC and develop a delivery strategy

for thermostabilized chABC in vivo.

To meet this goal, the following objectives were set:

1. To improve the thermal stability of chABC and develop a minimally invasive and

slow delivery system.

a. Determine ability of trehalose to improve thermostability of chABC in vitro.

b. Develop a hydrogel and lipid microtube mediated delivery scaffold and

evaluate functionality of post-released chABC in vitro.

2. To supply thermostabilized chABC in vivo by implanting a hydrogel-lipid microtube

delivery system with a topical delivery model and examine the functionality of

released chABC and the cellular responses after SCI.

a. Examine delivery efficiency of chABC by evaluating CSPG digestion two

after implantation.

3. To apply a combinatorial strategy using chABC and NT-3 to enhance therapeutic

effects and examine axonal sprouting/regeneration and functional recovery after SCI.

4
1.4. REFERENCES

Barritt AW, Davies M, Marchand F, Hartley R, Grist J, Yip P, McMahon SB, Bradbury EJ
(2006) Chondroitinase ABC promotes sprouting of intact and injured spinal systems
after spinal cord injury. J Neurosci 26:10856-10867.

Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon
SB (2002) Chondroitinase ABC promotes functional recovery after spinal cord injury.
Nature 416:636-640.

Fox K, Caterson B (2002) Neuroscience. Freeing the brain from the perineuronal net. Science
298:1187-1189.

Krekoski CA, Neubauer D, Zuo J, Muir D (2001) Axonal regeneration into acellular nerve
grafts is enhanced by degradation of chondroitin sulfate proteoglycan. J Neurosci
21:6206-6213.

Moon LD, Asher RA, Rhodes KE, Fawcett JW (2001) Regeneration of CNS axons back to
their target following treatment of adult rat brain with chondroitinase ABC. Nat
Neurosci 4:465-466.

Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L (2002) Reactivation of

ocular dominance plasticity in the adult visual cortex. Science 298:1248-1251.

Schwab ME, Bartholdi D (1996) Degeneration and regeneration of axons in the lesioned
spinal cord. Physiol Rev 76:319-370.

Yick LW, Wu W, So KF, Yip HK, Shum DK (2000) Chondroitinase ABC promotes axonal
regeneration of Clarke's neurons after spinal cord injury. Neuroreport 11:1063-1067.

5
 CHAPTER 2

RELEVANT BACKGROUND

Injuries trigger cellular and molecular signal cascades which upregulate inhibitors to

axonal outgrowth and result in regenerative failure. Inhibition could be caused by myelin-

associated inhibitors, immune-response molecules, glial scars, inhibitory extracellular matrix

(ECM) molecules, and the lack of trophic factors. One major class of growth inhibitors are

chondroitin sulfate proteoglycans (CSPGs) that accumulate around lesion site after SCI.

Studies have investigated CSPG-mediated inhibition to axonal regeneration.

Chondroitinase ABC (chABC) digests glycosaminoglycan chains on CSPGs and can

potentially overcome CSPG mediated inhibition and promote axonal sprouting/regeneration

when delivered into lesion sites. However, chABC loses its enzymatic activity rapidly at 37

ºC, necessitating the use of repeated injections or local infusions with a catheter and pump

for days to weeks to digest the CSPGs being produced continuously. Maintaining these

infusion systems is invasive and clinically problematic.

To overcome the limitations of chABC therapy for clinical application, the following

technical achievements must be met; stable bioactivity of chABC, a delivery system for

sustained local delivery in vivo, and a non-pump/catheter administration. In this chapter,

limitations that need to be overcome are described, and alternative methods and strategies to

solve these challenges are discussed.

6
2.1 CELLULAR AND MOLECULAR RESPONSES AFTER SCI

After injury to the central nervous system (CNS), the inflammation process is

triggered, a series of cellular and molecular responses are cascaded and eventually glial scars

are formed around the lesioned tissue (Fawcett and Asher, 1999). The failure of neurite

regrowth and permanent functional loss result from these cellular events. To begin,

macrophages and microglia, oligodendrocyte precursors, meningeal cells and astrocytes

migrate into the lesion site. These cells produce inhibitory molecules, such as myelin-

associated glycoprotein (MAG), CSPGs and other proteoglycans, free radicals, nitric oxide,

etc. The final form is a tightly interwoven glial scar formed around the lesioned site (Rudge

and Silver, 1990). Macrophages from the bloodstream and microglia from the surrounding

tissues are the first cells to arrive at the site, usually within a few hours after injury. They

remove myelin debris, produce and release cytokines, recruit oligodendrocyte precursors and

initiate reactive astrocytes. Inhibitory molecules are up-regulated and the injury site is

exposed to non-permissive environment for axonal regeneration. Myelin-derived growth

inhibitory proteins, such as NOGO, myelin associated glycoprotein (MAG), and

oligodendrocyte-myelin glycoprotein (OMgp), contribute to the failure of optic nerve

regeneration and spinal cord regeneration (Selles-Navarro et al., 2001). Oligodendrocyte

precursors migrate from the surrounding tissue after 3-5 days and myelin debris contains

myelin-associated inhibitory molecules: NOGO is present on the myelin surface and

collapses the neuronal growth cone (Bandtlow et al., 1993); MAG is produced by and present

in oligodendrocytes (Kastin and Pan, 2005); and OMgp is most recently identified and

inhibits neurite outgrowth (Kottis et al., 2002).

7
 If meningeal layers, which cover the CNS, are penetrated, meningeal cells migrate to

cover the exposed area of CNS and form a barrier to axon regeneration. Astrocytes are the

predominant and final structure of the astro-glial scar with ECM and ECM molecules. These

cells divide in reactive form, slowly migrate into the injured area, fill the space caused by

injury, and produce inhibitory molecules, such as chondroitin sulfate proteoglycans (CSPGs).

This astro-glial scar acts as a barrier that prevents axons from passing through and inhibits

axonal regeneration. CSPGs are over-expressed in extracellular matrix at the lesion site

where reactive astrocytes are present, and ultimately form astro-glial scars that act as barriers

to axonal outgrowth. CSPGs are generally accepted as potent inhibitory molecules of axon

growth in the adult CNS. Therefore, recent studies focus on alleviating CSPG-mediated

inhibition by delivering the enzyme chABC in vivo and to encourage axonal regeneration and

functional recovery (Moon et al., 2001; Bradbury et al., 2002; Barritt et al., 2006).

2.2 CSPG-MEDIATED INHIBITION AFTER SCI AND CURRENT TREATMENTS

It is known that axons of the CNS cannot regenerate to form functional connections

after injuries. However in the mid-1980s studies demonstrated that CNS neurons can

regenerate their axons over long distances when a favorable environment is provided (So and

Cho, 1989). Currently, many studies are investigating CSPG-mediated inhibition by

removing the inhibitory nature of CSPGs through enzyme-mediated modification. These

studies suggest that attenuating inhibitory effects of CSPG by chABC digestion enhances

axonal growth and functional recovery. chABC is a bacterial lyase produced from the

bactreum Proteus vulgaris that is able to digest CSPGs. The therapeutic significance of

chABC has been demonstrated in studies that delivered this enzyme to enhance nerve

8
regeneration (Bradbury et al., 2002; Barritt et al., 2006). However there are several

limitations to clinical treatment before implementation can occur.

2.2.1 CHONDROTIN SULFATE PROTEOGLYCANS

CSPGs consist of a core protein and glycosaminoglycan (GAG) chain that are

covalently linked to form a brush-like structure (Fig. 2.1). The GAGs are linked to the core

protein by a trisaccharide composed of two galactose and one xylose residue. GAG is made

of repeating disaccharides containing either two modifier sugars, glucuronic acid (GlcNAc)

or galactosamine (GalNAc) and an uronic acid (glucuronate or iduronate). These chondroitin

sugars can have over 100 individual sugar molecules and each can be sulfated in various

positions and quantities. Figure 2.2 shows that chondroitin sulfate glycosaminoglycans

(CSPG) have structural diversity (e.g. chondroitin-4-sulfate (∆glcA-β1,3-4S-galNAc),

chondroitin-6-sulfate (∆glcA-β1,3-6S-galNAc), chondroitin-2,6-sulfate (∆2S-glcA-β1,3-6S-

galNAc), dermatan-4-sulfate (∆iduA-α1,3-4S-galNAc), etc.) (Gilbert et al., 2005).

CSPGs are upregulated after injury in the CNS and become a major contributor to the

failure of axonal regeneration (Pindzola et al., 1993; McKeon et al., 1995; Fitch and Silver,

1997; Asher et al., 2000; Asher et al., 2002; Jones et al., 2003; Tang et al., 2003). CSPGs

also regulate neurons during development by defining barriers in CNS structures:

perineuronal nets (PNNs) (Matthews et al., 2002) in the brain and spinal cord, the roof plate,

a putative axon barrier, of the spinal cord and optic tectum (Snow et al., 1990a), and the

hippocampus (Wilson and Snow, 2000). Studies have shown that neurite outgrowth is

inhibited by CSPGs in vitro (Snow et al., 1990b; McKeon et al., 1991; Snow and Letourneau,

1992; Condic et al., 1999; Hynds and Snow, 1999; Snow et al., 2001; Johnson et al., 2002)

9
and in vivo after injury of optic nerve (Selles-Navarro et al., 2001) or the dorsal root entry

zone (Zhang et al., 2001). However, the precise contribution of different CSPGs to CSPG-

mediated inhibition in the CNS and the mechanism of cellular transduction of the inhibitory

signal mediated by CSPGs are not clear. Because CSPGs have a significant structural

diversity, the nature and profile of CSPG deposition patterns have been recently investigated

(Jones et al., 2003; Properzi et al., 2005). Specific CSPGs are differentially over-expressed in

astro-glial scar after injury and CSPGs have varying degrees of inhibition (Gilbert et al.,

2005).

After spinal cord injury, glial scars present a physical barrier and also produce

repulsive molecules such as CSPGs. This effect of CSPG-mediated inhibition can be

attenuated by applying chABC, which is an enzyme degrading CS-GAG (Snow and

Letourneau, 1992; Zuo et al., 1998; Krekoski et al., 2001). Therefore, enhanced axonal

regeneration can be achieved by removing CSPGs or the GAG side chains of CSPGs with

enzymes, such as chABC.

2.2.2 CHONDROITINASE ABC

chABC is one of a class chondroitinases and has a molecular weight of 120 to 145

kDa as determined by gel filtration (100 k Da in the SDS-PAGE). It is produced from

bacterium Proteus vulgarism and there is no mammalian equivalent (Ryan MJ, 1994).

chABC cleaves chondroitin, chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C-6-S),

dermatan sulfate (DS) and hyaluronan GAGs by β-elimination of 1,4-hexosaminidic bonds

into disaccharides and tetrasaccharides, yielding protein enriched core molecules with a

linkage of oligosaccharides (Oike et al., 1982).

10
Figure 2.1. Structure of chondroitin sulfate proteoglycan (CSPG). (A) versican,
a large CSPG belonging to the lectican family and (B) decorin, a small CSPG
of extracellular matrix.
(Figure modified from http://web.virginia.edu/Heidi/chapter9/chp9.htm)

11
 Figure 2.2. Schematic structure of various CSPGs and hyaluronan. Figure
from Gilbert et al., 2005.

The chABC is isolated from Proteus vulgaris contain two distinct eliminases, endolytic

(chABC I) and exolytic (chABC II) lyases (Hamai et al., 1997). chABC is commercially

available and the commonly used chondroitinase enzymes are provided by Seikagaku

Corporation (Japan) as “chondroitinase ABC (chABC I + chABC II)” and “chondroitinase

ABC, protease-free (chABC I)”. Usually the highly purified chABC, “chondroitinase ABC,

protease-free”, has been used for in vivo study and is used in this study.

chABC I is a 997 amino acid residue monomeric protein that degrades its substrates

to tetrasaccharides and disaccharides. The complete crystal structure of chABC I has been

12
determined to contain three domains: the N terminal, central, and C terminal domains. The

overall dimensions of chABC I is 115 Å × 70 Å × 55 Å and is folded into the three distinct

domains (Huang et al., 2003). This structure shows overall similarity to other GAG lyases

with additional domains on the N terminal. About 400 amino acid residues are in the C

terminal domain also shows a homology to chondroitinase AC (cAC; two domain) (Fethiere

et al., 1999) and bacterial hyaluronidases (Li et al., 2000). The role of this C-domain is not

clear, however, one loop in this domain acts as a substrate-binding site. The N terminal

domain consists of residues 25-234 and is the most flexible in chABC I compared to others.

The N-domain has a common topology to other ligand-binding domains, particularly the

carbohydrate-binding domains of xylanases and glucanases (Lo Conte et al., 2000). The

central domain (residues 235-617) contains 15 α-helices and is most likely the catalytic site.

The α-helix domain shows very similar sequence identity to the catalytic domains of cAC

and hyaluronidase. The active site of chABC I, as informed by superimposition with cAC

structure, has been investigated (Prabhakar et al., 2005a; Prabhakar et al., 2005b). However,

the active site has not been clearly defined and it is still unknown as to why chABC quickly

loses its enzymatic function at body temperature. In addition, a strategy has not been

developed to increase the enzymatic lifetime of chABC.

13
Figure 2.3. Ribbon drawing of chondroitinase ABC I (figure from
(Huang et al., 2003)); the N-terminal (green), middle catalytic (blue)
and C-terminal domain (yellow), from top to bottom.

14
 chABC has the widest substrate specificity; therefore, it has been used widely,

including as a therapeutic agent for SCI. Treatment with chABC has shown promising results

with various CNS applications, such as regeneration of ascending sensory neurons and

descending corticospinal tract axons in the spinal cord (Bradbury et al., 2002), sensory

neurons in the dorsal root entry zone (Steinmetz et al., 2005), and retinal ganglian cell axons

in the tectum (Tropea et al., 2003). Therefore, chABC offers a potential treatment strategy for

neural injury.

2.2.3 DELIVERY OF CHONDROITIANSE ABC FOR AXONAL REGENERATION

AND LIMITATIONS

chABC promotes axonal regeneration and functional recovery after CNS injury (Yick

et al., 2000; Moon et al., 2001; Bradbury et al., 2002; Yick et al., 2003; Chau et al., 2004)

and developmental disease (Pizzorusso et al., 2006). It promotes neurite sprouting in intact

and injured spinal cords (Barritt et al., 2006). Most groups deliver chABC via intrathecal

injection varying the infusion frequency from every other day to every other week, for time

periods ranging from 2 weeks up to 6 weeks (Chau et al., 2004; Caggiano et al., 2005; Houle

et al., 2006; Huang et al., 2006). Some groups inject chABC in combination with other

therapeutic agents (Tropea et al., 2003) or with transplantation, such as that of E14 fetal

spinal cords (Kim et al., 2006) or autologous peripheral nervous grafts (Houle et al., 2006), to

obtain a synergistic effect.

However, there are several limitations and difficulties to these methods. chABC is

thermally very sensitive at body temperatures, so after 1 hour of incubation at 37 ºC it

quickly loses 50% of its enzymatic activity (by Morgan-Elson reaction). It loses most of its

15
enzymatic function after a day. In addition, CSPGs are upregulated and accumulate in the

extracellular matrix around the injured site up to at least 2 weeks after the primary injury.

Therefore, most researchers continuously infuse chABC intrathecally through mini-

pumps/catheters to maintain a supply of fresh enzyme. Another limitation is the diffusion of

chABC. Most of these deliveries are performed intrathecally with a catheter, so it is hard for

chABC to diffuse into deep regions of the tissue. Because intrathecal delivery allows the

drug to flow through intrathecal space, the drug washes away quickly and does not last a long

time in the target area. Also, because of this drug diffusion, the concentration immediately

dilutes to significantly low concentrations, so high concentrations (from 2U/ml to 1000U/ml)

are needed to compensate. This is a problem for chABC. Because this enzyme is very

expensive, it is costly to apply therapeutically in high concentrations. As we can see, there

are several problems to overcome; specifically there is a compelling need to control chABC

delivery both spatially and temporally.

2.3 THERAPEUTIC STRATEGIES TO IMPROVE THERMAL STABILITY OF

PROTEINS

Thermal stabilization of protein has many practical applications in research and

industry. Since chemical reactions are generally faster at higher temperatures, enzymes,

which are stable at higher temperature, would yield a more efficient process (Schoemaker et

al., 2003; Unsworth et al., 2007). Also in the laboratory, thermally stable proteins/enzymes

are easier to store or handle. Many studies have been conducted to engineer thermostability

of proteins and there are two general strategies for protein engineering to improve the

thermal stability of proteins or enzymes (Bae et al., 2008): rational design and directed

16
evolution. Another strategy is to add cosolvents for modifying solvent environment (Cleland

and Wang, 1990).

2.3.1 PROTEIN ENGINEERING

In rational design, precise changes on amino acid sequence are designed from

detailed knowledge of the structure and function of the protein by using site-directed

mutation (Chen, 1999; Eijsink et al., 2004). It is generally inexpensive and easy, however the

detailed structure of a protein is often not available and even with the detailed information it

is difficult to predict the effect of mutation on protein functions. The other strategy, directed

evolution, involves generation of random mutations in the protein sequence, and does not

require information as to how the structure is related to function (Kuchner and Arnold, 1997;

Eijsink et al., 2005). This mimics natural evolution; random mutation is applied to a protein,

selected to pick desirable variants, and further rounds of mutation and selection are applied.

This directed evolution approach is hard to predict the results of stability and functionality,

and it requires an effort to narrow down best candidate. Random or rational

engineered/directed mutation is generated by screening a library of variants to produce a

stable variant. Many mutations that result in the most stable variant would have been difficult

to predict by rational design and the results of directed evolution are often better than rational

results. However these two techniques are not mutually exclusive and recently researchers

engineer proteins in combination of these methods to render desired changes (Eijsink et al.,

2004).

Enzyme stability can be improved by rational design, directed evolution or in

combination. Surface position is good candidate for optimizing protein stability (Martin et al.,

17
2002). Surface or near-surface interactions are important for protein stability. However, very

limited number of mutants can lead to an increase of stability and do not affect functionality.

Random method has been used to generate mutation and it can lead high stability, but some

of which are not easy to define rationally. Therefore, recently semi-rational method is

frequently used. To apply for our case, chABC, several charged residues could be selected

from the surface of the 3D structure (Prabhakar et al., 2005a; Prabhakar et al., 2005b), except

active residues, and randomly mutate these residues and find optimized mutants. The stability

of mutated chABC can be determined by 2D structure (circular dichroism) or 3D (X-ray

crystal structure), and the functionality by enzymatic activity assays, such as, SDS-PAGE,

western blotting, DMMB and HPLC method.

2.3.2 USAGE OF COSOLVENTS IN AQUEOUS SYSTEM

The other approach to stabilize protein is through addition of cosolvents. Cosolvents

can be broadly categorized into the following: sugars and polyols (sugar alcohol), amino acid,

amines, salts, polymers and surfactants. The level of stabilization by different cosolvents

varies for particular proteins. Therefore, a cosolvent that stabilizes one protein/enzyme may

not stabilize another (Hatti-Kaul and Mattiasson). Sugars (Carninci et al., 1998) and polyols

(Xie and Timasheff, 1997a) are the more commonly used nonspecific protein stabilizers.

Amino acids are also commonly used as stabilizer or osmolytes (Taneja and Ahmad, 1994;

Remmele et al., 1998). The cosolvent may affect both the structural stability and activity of

protein. Sugars and polyols always have been shown to enhance the thermal stability of

proteins, but some amino acids have been found to destabilize the protein. The effects of salts

also depend on pH of the medium and their chemical nature (Rishi et al., 1998). Therefore,

18
sugar or polyol are safe candidates as cosolvents. Often higher concentration of protein

increases its own stability, however high concentration could lead a chance of protein

aggregation. Surfactant is often used to reduce protein aggregation or protein surface

adsorption, however the concentration of surfactants need to be carefully decide to avoid side

effects (Faustino et al., 2009).

2.3.2.1 TREHALOSE AS A PROTEIN STABILIZER

Among various cosolvents, trehalose shows an exceptional improvement of thermal

stability of protein (Kaushik and Bhat, 2003). Trehalose accumulates dramatically during

heat shock and stationary phase in many organisms, then enhances thermo-tolerance and

reduces aggregation of denatured proteins. Therefore, trehalose is a good candidate as a

stabilizer for chABC. It has not been found in mammals, however this improves the tolerance

of mammalian cells to desiccation and cryopreservation in vitro. This approach would be

more convenient than the engineering protein.

Trehalose is found in nature as a disaccharide, two α-D-glucose molecules with the

alpha bond in a 1α→1 glycosidic linkage (α-D-glucopyranosyl(1→1)-α-D-glucopyranoside).

It is commonly used as a sweetener in food and as a cryopreservation additive. It is also

known as an exceptional protein stabilizer, providing protection to biological materials

during dehydration and desiccation (Sampedro et al., 1998). It promotes survival under

extreme heat by stabilizing proteins in order to retain their conformation and suppresses the

aggregation of denatured proteins (Singer and Lindquist, 1998). Further, trehalose stabilizes

labile proteins during lyophilization (Zhang et al., 2009), protects enzymatic activity, such as

19
with RNase A, lysozyme, cytochromes during exposure to high temperatures in solution, and

in a freeze-dried state (Kaushik and Bhat, 2003).

Figure 2.4. Structure of trehalose.

2.3.2.2 THE POSSIBLE MECHANISMS OF TREHALOSE STABILIZATION OF

CHABC

As described previously, trehalose has unique properties with exceptional ability to

protect biological materials under extreme conditions (Cottone et al., 2005; Hedoux et al.,

2006). Many studies have been conducted to understand the mechanism of trehalose and

possible mechanisms have been proposed, however the molecular mechanism of trehalose

stabilization is poorly understood (Hedoux et al., 2009). There are several hypotheses to

explain the mechanism of trehalose efficiency: 1) water replacement model (Crowe et al.,

1984), 2) preferential hydration hypothesis (Arakawa and Timasheff, 1983), 3) vitrification

of solutions (Green et al., 1989) and 4) the influence on the water tetrahedral hydrogen-bond

network (Branca et al., 1999). However, the molecular mechanism probably depends on the

nature of applied stresses.

20
 The preferential interaction theory was proposed by Arakawa and Timasheff in 1983.

They observed that bovine serum albumin and lysozyme were preferentially hydrated in

amino acids. Preferential interaction is also stated as preferential binding of the cosolvent or

preferential exclusion (preferential hydration). The cosolvent chemical potential perturbation

by the protein is the driving force of preferential interaction, which causes change of the

amount of water in contact with protein (Timasheff, 2002). Studies showed that trehalose

stabilizes the folded structure of proteins in solution due to greater preferential hydration of

the unfolded state compared to the native state (Xie and Timasheff, 1997b). Therefore, the

mechanism seems to be opposite from the stabilization mechanism in the dried state, which is

water replacement. Sugars generally protect proteins from dehydration by preventing the

decrease of spacing through hydrogen bonding to the dried protein surface, serving as a water

substitute (Carpenter et al., 1993). Like this, the mechanism of stabilization can be different

under different applied stress conditions.

The primary mechanism by which trehalose is likely to affect the stability of the

proteins is by increasing the surface tension of water around the proteins due to hydrogen

bond formation between the hydroxyl groups of trehalose and water. In solution, trehalose

stabilizes RNase A by increasing the surface tension of the trehalose solution, leading to the

preferential hydration of the protein (Xie and Timasheff, 1997a). Surface tension of trehalose

solutions increases linearly with increasing trehalose concentration (Kita et al., 1994), and

there is a strong correlation between surface tension and increase in Tm (Jai et al., 2003).

Increasing solvent surface tension necessitates more energy to form a cavity in order to

increase the surface area of proteins upon denaturation. In fact, other protein stabilizers, such

as polyols and carboxylic salts (Kaushik and Bhat, 1999) show a correlation between surface

21
tension increase and enhanced protein thermal stability. Studies also have shown that the

dynamic fluctuation of polar side chains at the solvent-protein interface is reduced in the

presence of trehalose (Hedoux et al., 2009). The increased surface tension or the limited

exposure of hydrophobic groups to the water molecules leads to preferential hydration,

resulting in the stabilization of the tertiary structure of protein (Timasheff, 2002).

2.3.3 PROTEIN STABILIZATION BY PEGYLATION

PEGylation describes a process of covalent conjugation with polyethylene glycol

(PEG) polymer chains to biological molecules. PEG (Figure 2.5) manufactured by

polymerization of ethylene oxide with water, ethylene glycol or ethylene glycol oligomers

and various molecular weights of PEG can be prepared by modulation of the polymerization

reaction. In 1970s, Aluchowski et al. studied a pioneering method of PEG conjugation

(Abuchowski et al., 1977). In the study, methoxypolyethylene glycol was covalently attached

to bovine serum albumin and it lost its immunogenicity and the potential of PEG conjugation.

After this successful study, a number of studies followed and PEG has been applied to

several biological therapies and success have been achieved in parallel with improvements of

the PEG properties (Veronese and Mero, 2008). PEG is a biologically favorable molecule, a

non-toxic and non-immunogenic molecules approved by the US FDA for internal use.

PEGylation has been well established in the clinic and there are many PEGylated

pharmaceuticals on the market, such as Pegasys (PEGylated interferon alpha), Oncaspar

(PEGyleated L-asparaginase), Neulasta (PEGylated granulocyte colony-stimulating factor),

Doxil/Caelyx (PEGylated liposome containing doxorubicin), etc (Veronese and Mero, 2008).

22
 Figure 2.5 Poly(ethylene glycol)

Proteins/enzymes are delicate molecules and easily denatured or deactivated.

PEGylation is can be applied to these therapeutic agents to modify or improve their stability,

degradation by proteases or immunogenicity, and degree of renal excertion. The covalently

attached PEG chains change the physical and chemical properties of the agents. Several PEG

chains are attached on the surface of agents and result in increasing the molecular weight and

creating water cloud surrounding the PEGylated agents by hydrogen bond formation between

ether-oxygen in PEG (Figure 2.6). The modification improves pharmacokinetics of drugs:

shielding the PEGylated agents from the host‟s immune system reduces immunogenicity and

antigenicity and increases stability. It also provides high solubility in both aqueous and

organic solvent, high mobility in solution, high hydration increasing hydrodynamic size in

solution, and increases of the retention time in blood (Israelachvili, 1997). Due to the

pharmacokinetic benefits of PEGylation, therapeutic proteins, peptides and antibody

fragments have been PEGylated for several drug delivery applications (Harris and Chess,

2003).

23
 Figure 2.6 Schematic of PEGylated protein. The circles represent the
water cloud recruited by the ether-oxygen groups of the PEG polymer.
Figure from Veronese and Mero, 2008.

Hydroxyl group of the PEG terminal can react with the various protein amino acid

residues. Since amino groups are present in every protein, generally at the surface and

exposed to the solvent, it is the most exploited residue for PEGylation by alkylation or

acylation. Amino group modification can negatively affect surface properties of the protein

and in some cases the modification decreases enzymatic/biological activity (Banci et al.,

1990; Greenwald et al., 2003). Modifying cysteine thiol residue by formation of thio-ethers

or disulfides allows site-specific conjugation. Generally cysteine residues are involved in

catalysis or disulfide bridges, so available free cysteine residues are rarely present. Therefore,

sometimes this residue is genetically introduced in a desirable position for convenient

applications (Kopchick et al., 2002). Disulfide-linked PEG conjugation can be a reversible

modification and it allows a releasable PEGylation under mild reducing conditions

(Woghiren et al., 1993). Similarly customized triggering moiety by enzymatic reaction,

hydrolysis, or linker self-immolation cleaves the link and releases PEGylation, regenerating

native agents and preserving its bioactivity. Recent reports demonstrate that the customized

linkers of cytokines, peptide hormones, enzymes or receptor proteins are released under

24
physiological conditions and at specific and therapeutically useful rates (Filpula and Zhao,

2008).

Other amino acid residues, such as arginine (Veronese and Mero, 2008), glutamine,

the alcohol group of serine and threonine, and the phenolic group of tyrosine (Orsatti and

Veronese, 1999) are usable sites for PEG conjugation. For glutamine residue conjugation, the

enzyme, transglutaminase, is used because there is no chemical method (Sato et al., 1996)

and this glutamine-PEGylation is very specific (Fontana et al., 2008). However, because of a

lack of specificity (arginine) and harsh reaction conditions (serine and theronine) other

methods still need to be improved for therapeutic applications.

Since covalent binding is involved in PEGylation, and several PEG chains are usually

linked on the surface, the size and conformation of the agents are changed. Also, there is a

possibility that conjugated PEG polymers can block the binding sites and active sites

resulting in loss or damage of the agent‟s bioactivity. Therefore, the mass, number of chains

and link positions need to be investigated for each case to maintain its bioactivity. To avoid

these complications, genetically engineered proteins can be used: a genetic variant of amino

acids is inserted at a site far from biologically active site and used for a residue to PEG

conjugation (Kopchick et al., 2002).

PEGylation has many advantages and some disadvantages to be used as a protein

stabilizer. In our case with chABC, it would not be an ideal method for several reasons.

Linked PEG chains can mask catalytic site of chABC, however exact positions of the site are

still not clear and the mechanism of deactivation of chABC enzymatic activity is also

unknown. Therefore, it is not easy to design a relevant PEG polymer conjugation for chABC

to thermostabilize its enzymatic activity. Additionally, chABC is a relatively large molecule

25
and the increased size of chABC with PEGylation will result in limited diffusion through the

nerve tissue. Therefore, we decided to use cosolvent in this study.

2.4 THERAPEUTIC STRATEGIES FOR CONTROLLED RELEASE OF AGENTS BY

DELIVERY CARRIERS

The formulation of macromolecular agents, such as proteins, enzymes, and DNA in

drug delivery systems is widely investigated due to the increased needs as therapeutics.

Various carriers have been used for the efficient delivery of agents for different therapeutic

purposes. For slow and local delivery of chABC after SCI, it is important to control

temporally and spatially the amount of protein delivered over a period of time. Also, the

delivery vehicle is able to maintain bioactivity of the agents over the period of time. The

implanted vehicle itself and by-product of degraded vehicle should not induce significantly

increase of inflammation response or aggravation of immune responses. Currently major

delivery method of chABC for clinical and in vivo application to treat SCI is intrathecal

injection through the implanted catheter with mini-pump or syringe (Bradbury et al., 2002;

Barritt et al., 2006). This delivery system consists of two parts: 1) an infusion pump or an

external catheter end implanted under the skin on the back of the animal, and 2) catheter

inserted in the lesion site. However the internal catheter end is chronically implanted, and

human cases have reported that intrathecal catheter-tip causes inflammatory mass (Peng and

Massicotte, 2004), and surgical site infection (Burgher et al., 2007). Also an insoluble gelatin

sponge and gelfoam containing chABC was used to treat SCI (Yick et al., 2003). In this

study, we investigated a carrier-based drug delivery.

26
2.4.1 POLYMER NANOPARTICLE

Various delivery vehicles are used to deliver drugs into the CNS and PNS, such as

liposomes, polymer nanoparticles and lipid microtubules. In order to locally deliver drugs for

spinal cord treatment, PLGA nanoparticles and lipid microtubules could be considered for

sustained delivery. Our laboratory has used both vehicles for in vivo sustained drug delivery.

PLGA is a biocompatible and biodegradable polymer and is approved by US Food and Drug

Administration for human use. PLGA nanoparticle have been used for sustain release of

encapsulated agents (Shive and Anderson, 1997). The double emulsion method is used to

make PLGA nanoparticles and is simple and takes a relatively short time, a total of 3 days.

Additionally, it is easy to store in nanoparticle powder form at -80 ºC after lyophilizing.

However during the double emulsion procedure, the releasing agent, such as protein, can be

exposed to organic solvents (ex. Dichloromethane) and high temperatures. Without co-

encapsulation of acid-neutralizing base, the pH is predominantly below pH 5.8 (detection

limit in this paper) (Li et al., 2005). Therefore proteins undergo physical denaturation and

chemical degradation during fabrication (Sah et al., 1999; Kim et al., 1999) and

unpredictable release profiles often occur, such as a burst effect or incomplete release (Kim

et al., 1999). Only a few proteins have shown the ability of controlled delivery from PLGA

spheres (Kim et al., 2005).

2.4.2 LIPID MICROTUBES

Lipid microtubes are also capable of the slow release of protein or bio-agents for in

vivo application. It spontaneously forms hollow and open-ended cylinders (Schnur, 1993)

with 0.5 µm of the average inner diameter and the lengths of the cylinders depend on the

27
cooling rate (Thomas et al., 1995; Meilander et al., 2001). Microtubes have a high aspect

ratio and it allows for a large storage volume and they are stable in physiological medium at

37 ºC (Spargo et al., 1995) for prolonged a period of time. The fabrication process takes a

relatively long time, a total of 15 days, and microtubes need to be prepared freshly. However,

there is no toxic procedure for proteins, which could cause denaturation or degradation,

during the fabrication process. Microtubes show a stable and continuous release profile and

could be used as sustained delivery vehicles for proteins, such as neurotrophic factors (Jain et

al., 2006), and even nucleic acids (Meilander et al., 2003). It is also easy to combine with

hydrogels to support a scaffold for in vivo application and non-inflammatory (Rudolph et al.,

1992; Meilander et al., 2001). Lipid microtubes are injectable either by themselves, or when

embedded in thermo-reversible hydrogels as reported in Jain et al. (2006) for localization.

The study showed that an agarose hydrogel scaffold embedded by BDNF loading lipid

microtubes was implanted into a spinal cord cavity and led to the reduction of the

inflammatory response and enhanced axonal infiltration into the scaffold.

The molecules are released through the ends of microtubes, and a mathematical

model of release profile of proteins has been developed previously in our laboratory

(Meilander et al., 2004). The release of proteins can be predicted with the molecular weight

and protein concentration. The slow release of agents can be modified by controlling the

length of microtube, type of gel, and gel concentration for desire delivered amount of agent

for different application.

28
2.5 PHARMACOLOGICAL STRATEGIES FOR SCI

2.5.1 NEUROPROTECTIVE THERAPY TO TREAT ACUTE SCI

There are limited therapies for SCI and no effective treatment to reduce damage and

to promote functional recovery (Martinon and Ibarra, 2008). Methylprednisolone (MP),

which is a synthetic cortico-steroid and typically used for anti-inflammatory effect, has been

used as a drug for acute SCI in humans for three decades with limited clinical support, and it

is the only available drug for acute SCI in human. However it is based in large part on

physiological hypotheses and its beneficial effect on the neurological recovery of patients has

not been clearly proven (Hugenholtz, 2003). The effect of MP administration in patients with

acute spinal cord injury was clinically examined by National Acute Spinal Cord Injury Study

I, II and III (NASCIS) (Bracken et al., 1984; Bracken et al., 1985; Bracken et al., 1990;

Bracken et al., 1992; Bracken et al., 1997; Bracken et al., 1998). In NASCIS II, a patient sub-

group received a 24 hour high-dose infusion of MP within 8 hours following acute SCI

showed improved neurological recovery (Bracken et al., 1990). Therefore within 8 hours

after injury and high-dose infusion (typically 30 mg/kg bolus injection and 5.4 mg/kg/h

following injection over 23 hours) has been an implied standard for clinical treatment. The

high-dose of MP causes several side effects, such as impaired lung capacity and the higher

incidence of sepsis and pneumonia. Compelling evidence of its efficacy is not yet conclusive

(Gerndt et al., 1997). To minimize the side effects related to systemic delivery, localized

delivery methods have been developed: previously, our group developed a sustained and

local delivery method. MP was delivered on the top of the lesion site by agarose hydrogel

delivery system embedded with biodegradable polymer nanoparticles and showed effective

diffusion through the spinal nerve tissue and significantly reduced early inflammation

29
(Chvatal et al., 2008; Kim et al., 2009). However, still there is the confusion with MP utility

for acute SCI and MP should be used with caution, particularly if infusion goes longer than

24 hours.

A wide range of other pharmacological treatments have also been evaluated and in

some cases have shown potential with promising results. Cyclooxygenase (COX) is an

enzyme that is involved in prostanoids formation. Pharmacological COX inhibitor acts as an

anti-inflammatory agent reducing inflammation and pain, and some selective (indomethacin;

(Pantovic et al., 2005)) or non-selective (NS-398 to COX-2; (Hains et al., 2001)) COX

inhibitors promoted neuroprotection. Immunophilins (IPs) are peptidyl-prolyl cis-trans

isomerases and some IPs are receptor for immunosuppressive drugs such as cyclosporine A

and FK506 (Sosa et al., 2005). Drugs inhibits the activity of calcineurin, which is a protein

phosphatase and activates the T cells of the immune system, and the drug binding can

promote neuroprotective effect and results in inducing neuroregeneration (Ibarra and Diaz-

Ruiz, 2006).

Oxygen radical-induced lipid peroxidation (LP) plays an important detrimental role in

acute CNS injury. Therefore, several therapeutic strategies have been applied to diminish its

effects (Hall et al., 1992). As previously mentioned, MP has been used for human SCI and

lazaroids, 21-aminosteroids, also show significant antioxidant effects without the same side

effects of MP (Hall and Springer, 2004). Tirilazad, one of lazaroids, was also examined in

the NACIS III and tirilazad treated patients showed slightly better neurological recovery, but

not significantly higher than those treated with MP (Bracken et al., 1997). Therefore, there is

a possibility for the use of tirilazad in humans bearing FDA approval.

30
 Calpains are a family of calcium-dependent, non-lysosomal cysteine proteases.

Hyperactivation of calpains follows traumatic brain injury or spinal cord injury due to Ca2+

influx and it leads irreversible cell damages such as breakdown of cytoskeleton and plasma

membrane and damage of ion channels, cell adhesion molecules and surface receptors.

Therefore, calpain can cause neural cell apoptosis after SCI (Ray et al., 2003). Highly

specific inhibitor to calpain for therapeutic use has been investigated such as E-64-d (Zhang

et al., 2003) and leupeptin (Momeni and Kanje, 2006), and these inhibitors demonstrated

neuprotective ability in models of SCI.

Besides those therapeutic strategies, there are a number of pharmacological therapy

targets, such as apoptosis inhibitors, steroid hormone and soldium channel blockers, and

NMDA and AMPA-Kainate receptor antagonists (Martinon and Ibarra, 2008).

2.5.2 NERVE GROWTH FACTORS: NEW POSSIBLE THERAPEUTIC STRATEGY

Several neurotrophic factors induce neuroprotection and promote axonal outgrowth

and functional recovery in traumatic injuries to the CNS. Neurotrophic factors that play an

important role in the survival, development and function of neurons, include brain-derived

neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin three (NT-3) and

neurotrophin-4/5 (NT-4/5). They belong to a class of growth factors and secreted proteins.

BDNF was originally found in the brain, but is also found in the peripheral nervous

system. It helps to support the survival of existing neurons and encourages the regrowth and

the differentiation of neurons and synapses (Waterhouse and Xu, 2009). Therefore, BDNF

has been used for treatment after CNS injuries to improve motor and sensory neuronal

survival and outgrowth (Schmidt and Leach, 2003).

31
 NT-3 is in the NGF-family of neurotrophins and binds three receptors: the receptor

tyrosine kinase neurotrophin receptors (TrkC and TrkB) and low affinity nerve growth factor

receptor (LNGFR). It helps to support the survival and differentiation of existing neurons,

and encourages the growth and differentiation of new neurons and synapses. NT-3 leads to

branching and elaboration of sensory endings (Krimm et al., 2004), promotes nerve

regeneration and sensory improvement (Sahenk et al., 2005) and acts as a survival factor for

adult sensory neurons (Ljungberg et al., 1999). Since the sensory pathway runs along the

dorsal column of spinal cord and a dorsal over hemisection was used in this study, NT-3 was

chosen for delivery into spinal cord to encourage nerve survival and outgrowth after the

injury.

For axonal regeneration through the lesion site after SCI, a balance between a

permissive and favorable environment for the axons in the lesioned area is important.

Therefore, in this study, a more permissive environment was achieved by degrading the

inhibitory molecules CSPGs with chABC, while an improvement of regeneration ability was

achieved by the supporting neurotrophic factor, NT-3.

2.6 CONCLUSIONS

Many researchers have shown that strategies delivering chABC alone or in

combination with other therapeutic agents give promising results after SCI, although there

are some limitations and difficulties. Therefore, there is a need to develop an alternative

method to control the release of chABC temporally and spatially in vivo. In this study, we

improved the thermal stability of chABC and developed a sustained delivery scaffold for

topical delivery model combining 1) the chABC improved thermal stability by introducing

32
trehalose; 2) a temporal control method using lipid microtubules for long-term and

continuous slow release; and 3) a spatial control method utilizing agarose gel to fabricate a

scaffold for local delivery to the lesion site via implantation.

This chapter briefly summarizes the background to design and develop strategies for

chABC or neurotrophic factor mediated treatment for SCI, as described above.

33
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44
 CHAPTER 3

IMPROVEMENT OF THERMOSTABILITY OF CHONDROITINASE ABC

ENZYMATIC ACTIVITY AND DEVELOPMENT OF LIPID MICROTUBE AND

HYDROGEL MEDIATED DELIVERY SYSTEM

(Partially published with R.J. McKeon and R.V. Bellamkonda, Proceedings of the National

Academy of Sciences, 2009)

Spinal cord injury (SCI) triggers cellular and molecular signal cascades which,

amongst other things, induce growth cone inhibitors and result in regenerative failure.

Chondroitin sulfate proteoglycans (CSPGs) are upregulated and accumulate around lesion

sites after SCI and are major inhibitors of axonal regeneration. To overcome the inhibitory

effect of CSPGs, modification or digestion of CSPGs has been explored. It has been reported

that chondroitinase ABC (chABC) can digest glycosaminoglycan chains on CSPGs and

enhance regeneration when delivered into lesion sites (Bradbury et al., 2002). However,

chABC has a crucial limitation; it is thermally sensitive and at body temperature, 37ºC, its

enzymatic activity is significantly attenuated within 72 hours (Tester et al., 2007). This

necessitates the use of multiple or continuous infusions with a pump to maintain enzymatic

functionality for periods as long as two weeks. However, maintaining these infusion systems

is invasive and clinically problematic. Here, to overcome the current limitations, we report: 1)

improvement of the thermal stability of chABC, and 2) development of a minimally invasive

strategy for delivery of chABC in vivo over a period of 2 weeks or longer. By adding a

protein stabilizer, trehalose, the thermostability of chABC was improved and chABC

45
maintained its enzymatic activity for 4 weeks. Enzymatic activity and conformational change

were assayed by functional and structural tests such as dimethylmethylene blue staining of

digested CSPGs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and circular

dichroism. A scaffold consisting of lipid microtubules and agarose gel was used for sustained

and spatially controlled release of thermally stabilized chABC in vitro. chABC released from

the gel-microtubule scaffold showed enzymatic activity for 2 weeks. These results have

important implications for strategies that aim to digest CSPGs as a means of reducing growth

cone inhibition after SCI.

3.1 INTRODUCTION

Physical damage to the adult central nervous system (CNS) often leads to permanent

functional loss due to the inability of mature axons to regenerate. A major impediment to

regeneration is the formation of astro-glial scar tissue at the lesion site along with a number

of myelin associated inhibitory moieties. After injury to the CNS, an inflammation process is

triggered that includes a cascade of cellular and molecular responses occurs and a glial scar is

formed around the lesioned tissue (Fawcett and Asher, 1999). Macrophages, microglia,

oligodendrocyte precursors, meningeal cells and astrocytes migrate into the lesion site and

produce inhibitory molecules, such as myelin-associated glycoprotein (MAG), CSPGs, free

radicals, nitric oxide, etc. The final „product‟ is a tightly interwoven glial scar formed around

the lesioned site composed primarily of CSPGs and reactive astrocytes.

chABC has been shown to promote axonal growth and functional recovery in a

number of models including spinal cord injury (Bradbury et al., 2002) and monocular

deprivation (Pizzorusso et al., 2006). It promotes axonal sprouting in both intact and injured

46
spinal cords (Barritt et al., 2006), perhaps via its action on perineural nets. Currently, chABC

is typically delivered via intrathecal injection, with infusion frequency varying from every

other day to every other week, and for time periods ranging from 2 weeks up to 6 weeks

(Chau et al., 2004; Caggiano et al., 2005; Houle et al., 2006; Huang et al., 2006). Some

groups inject chABC in combination with other therapeutic agents (Tropea et al., 2003) or in

combination with cell transplantation, such as cells from E14 fetal spinal cords (Kim et al.,

2006) or autologous peripheral nerve grafts (Houle et al., 2006), to obtain a synergistic effect.

However, there are several limitations to using chABC in vivo. ChABC is thermally

sensitive at body temperature, and loses 50% of its enzymatic activity after 1 hour of

incubation at 37 ºC (by Morgan-Elson reaction; Seikagaku, Japan). Most of its enzymatic

function is lost within 3-5 days (Tester et al., 2007). Generally, CSPGs are upregulated and

accumulate in the lesion site for at least 2 weeks after the primary injury. Therefore for

chABC to remove CSPGs, it would need to be delivered for at least two weeks intrathecally

through pumps to provide a „fresh‟ supply of enzyme. Another limitation is that the diffusion

of chABC into deep regions of the cord is limited when delivered intrathecally. Because of

this delivery method the drug flows through intrathecal space and washes away quickly,

necessitating high concentrations (from 2U/ml to 1000U/ml) compensate. Therefore, there is

a compelling need to control chABC delivery both spatially and temporally.

To overcome these limitations, trehalose was introduced as a stabilizer to retain the

enzymatic functionality of chABC. Trehalose is a superior stabilizer among sugars or polyols

and trehalose could give a longer lifetime to chABC. The concentration of trehalose was

decided after in vitro experiments with various concentrations of trehalose. Second, to

develop a sustained and local delivery system for providing bioactive agents, an agarose

47
hydrogel and lipid microtube was used. With the agarose hydrogel and lipid microtube

mediated delivery system, the release of the drug can be temporally and spatially controlled

by the combination of the concentration or type of agarose gel and the length of the lipid

microtube (Meilander et al., 2001). Agarose gel was used to construct an implantable

delivery carrier in vivo embedded with either lipid microtubes (Jain et al., 2006) or polymer

nanoparticles (Chvatal et al., 2008; Kim et al., 2009). This is referred to as the „hydrogel-

microtube delivery system‟. Using both trehalose and the hydrogel-microtube delivery

system, a sustained and local release of chABC and neurotrophine three (NT-3) was achieved.

In this study, we improved the thermal stability of chABC and developed agarose gel

scaffolds for spatio-temporal controlled delivery. The in vitro experiments performed

determined whether trehalose can be used as a protein stabilizer to improve the thermal

stability of chABC, and ultimately increase the lifetime of enzymatic activity at body

temperature. In addition, lipid microtubes and an agarose hydrogel were introduced to a

develop delivery scaffold and subsequently examined as a drug delivery system for spatio-

temporally controlled release in vitro. Last, we showed an alternative approach that will

allow us to apply chABC as a therapeutic agent in vivo, as described later in chapter 4 and 5.

3.2 MATERIALS AND METHODS

3.2.1 ENZYMATIC ACTIVITY ASSAY WITH SDS-PAGE

Because of its exceptional ability to stabilize and maintain the enzymatic activity of a

number of proteins, trehalose was used to stabilize chABC at body temperature (37 ºC)

(Kaushik and Bhat, 2003). To determine the proper concentration of trehalose for stabilizing

the enzymatic function of chABC, various concentrations of trehalose, from 20 mM to 1 M,

48
were tested for their ability to digest the GAG chains of a specific CSPG, decorin, which

consists of a core protein and a CS-GAG chain. Various concentrations of trehalose (20, 50,

100, 250, 500 mM and 1 M) were prepared in 1X phosphate buffered saline (PBS). chABC

(2U/0.5ml; 250ng) was mixed with these trehalose concentrations and each mixture was

incubated for 1, 2, 3 or 4 weeks in a 37 ºC water bath. After co-incubating for different time

durations of trehalose and chABC at 37 ºC, 10 µl of decorin (5 µg) was added, incubated at

37 ºC for 4 additional hours and the resultant products were analyzed with sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Penicillinase (P‟ase; 250 ng) was

used as a control enzyme.

When intact, decorin appears as a large molecular weight smear on an SDS-PAGE gel.

A tighter and lower molecular weight decorin band after incubation with chABC would

indicate preservation of chABC enzymatic activity. chABC without trehalose was used as a

negative control and was incubated at 37 ºC for the same incubation durations, mixed with

decorin, and incubated at 37 ºC for an additional 4 hours. After incubating, the mixtures were

diluted 1:1 with Tris-SDS sample buffer containing 5% β-mercaptoethanol and were further

incubated for 4 minutes at 95 ºC to denature the protein. The gel electrode assembly was

placed in an electrophoresis chamber (BIORAD) and the chamber was filled with Tris

running buffer. 20 µl of the prepared samples was loaded into each lane of SDS gradient gel

(4% - 15%; BIORAD) and run at 200 volts for 1 hour.

Silver staining was conducted to visualize separated proteins. Because the enzymatic

thermostability of chABC depends on lot and time (Tester et al., 2007), all experiments in

this study were performed with chABC from the same lot.

49
3.2.2 SILVER STAINING PROCEDURE

After running on SDS-PAGE, the gel was fixed in a solution containing methanol,

acetic acid, and water in a 45:5:50 ratio for 90 min and washed with water for 20 min twice.

The gel was washed with 0.02 sodium thiosulfate (Na2S2O3·5H2O) for 3 min and with

water for 30 sec twice, then washed with 0.1 % silver nitrate for 30 min and with water for

30 sec. A developing solution containing 2.5 % sodium carbonate/ 0.02% formaldehyde was

added. The gel was developed for 2-4 min and protein bands generally appeared within 30

sec. The developing solution was removed before overdevelopment and a solution containing

1 % acetic acid was added to quench the reaction. After rinsing with water, the gel was

imaged on the BIORAD ChemiDoc XRS HQ in the epi-white mode.

3.2.3 ENZYMATIC ACTIVITY ASSAY WITH DMMB

To further characterize stability of chABC, a dimethylmethylene blue (DMMB) assay

was used to quantify CS-GAGs that remained after digestion of decorin with chABC. Diluted

chABC (40mU) in 1X PBS (200µl) was mixed with 1 M of trehalose and incubated at 37 ºC

to subject it to thermal stress. 10 µl of incubated chABC solution was sampled for analysis

on day 1, 3, 7 and 14. The sampled chABC/trehalose was then mixed with 10µg of decorin

and incubated at 37 ºC for 10 minutes. Next, the DMMB reagent was added and absorbance

was measured on a Microplate Reader (BIO-TEK Instruments, Inc.). The same concentration

of chABC/trehalose without added decorin was measured as the background on the same

plate and the measured background absorbance was subtracted from absorbance of the other

samples. chABC in 1X PBS without trehalose was prepared to determine the degree to which

the enzymatic activity of chABC is improved by adding trehalose. The absorbance of decorin

50
digested by fresh chABC was considered to represent 100% of enzymatic activity. The

percentage of sample absorbance relative to this standard was calculated and a deactivation

curve was plotted. The absorbance of trehalose in 1X PBS at the same concentration as above

was measured to make sure that trehalose does not interfere DMMB results.

3.2.4 FABRICATION OF LIPID MICROTUBES

Hollow and open-ended lipid microtubes (Fig. 3.1.) were fabricated using 1,2-bis-

(triscosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC, Avanti Polar Lipids,

Alabaster, AL) as previously described (Meilander et al., 2001; Meilander et al., 2003).

Briefly, the lipid was dissolved in 70% ethanol at a concentration of 1 mg/ml. The solution

was slowly cooled from 55 ºC to 21 ºC, heated to 33 ºC and cooled again to 20 ºC by a

programmed water bath (Thermo NESLAB, Portsmouth, NH) for 48 hours. During this

cooling procedure, the microtubes self-assembled into hollow structures (Spargo et al., 1995;

Meilander et al., 2001). The average length of microtube can be controlled by modifying the

cooling process (Lando et al., 1990; Thomas et al., 1995). After 2 weeks of incubation at

room temperature, 50 mM (18.9 mg/ml) of trehalose was added to the microtube solution,

mixed gently and incubated overnight to support the stability of the lipid microtube structure.

The microtube solution was centrifuged for 5 min at 3000 rpm, dried overnight in a

biological hood for complete lyophilization. Microtubes were loaded with chABC, NT-3 or

P‟ase in buffer, 1 M trehalose in 1X PBS, and incubated overnight at 4 ºC to allow for full

rehydration. Microtubes were transferred to a new tube and extra buffer, 1 X PBS, was added

to wash out left-over agents outside.

51
 Figure 3.1. Micrograph of lipid microtubes formed from DC8,9PC lipid in
bright field microscopy, scale bar is 50 µm. Figure from Meilander et al.,
2001.

3.2.5 PREPARATION OF AGENT-LOADED LIPID MICROTUBES

The loading concentration of the drug was calculated based on the loading efficiency.

The loading efficiency was defined as the ratio of the entire internal volume of the

microtubes to the volume of the drug solution added to lyophilized microtubes (Meilander et

al., 2001). Figure 3.2 shows a histogram of the lengths of microtubes used in this study. The

average length was about 37 µm (0.5 µm of inner diameter) and the total number of

microtubes from 1 mg of lipid was approximately 1 × 108. Therefore, the total inside volume

of microtubes yielded from 1 mg of lipid was 0.75 µl. To increase the loading efficiency, we

tried to increase the internal volume of lipid microtubes by using more lipid solution,

52
increasing the surface area of lyophilized microtubes for more efficient rehydrating with the

solution of interest, and decreasing the loaded volume of solution of interest.

500
Number of Microtubes

400

300

200

100

Length of microtubes (µm)

Figure 3.2. Histogram of microtube length distribution. The average length

is about 37 µm, total number of microtubes from 1 mg of lipid is
approximately 1 × 108 and total inside volume of microtubes from 1 mg of
lipid is 0.75 µl.

3.2.6 ENZYAMTIC ACTIVITY ASSAY OF POST-RELEASED CHONDROTINASE

ABC FROM LIPID MICROTUBES/HYDROGEL-MICROTUBE DELIVERY

SCAFFOLD

SDS-PAGE was used to examine any potential adverse effects of the lipid microtubes

on chABC as a carrier for sustained release, such as loss of enzymatic activity. The DMMB

53
assay was used to characterize the release profile of chABC from microtubes. After preparing

microtubes loaded with chABC (~20 mU) and 1 M trehalose, the microtubes were co-

incubated with decorin (5 µg/200 µl). After 4 hours at 37 ºC, the co-incubated samples were

centrifuged and the supernatant was analyzed by SDS-PAGE as described in the section 3.2.1.

The enzymatic activity of released chABC from the hydrogel-microtube delivery

system was characterized via DMMB assay. In order to physically maintain

chABC/trehalose/microtubes in place in vivo, SeaPlaque® agarose (Cambrex), an agarose

hydrogel, was dissolved in 1X PBS at a concentration of 1.2% (w/v) (Jain et al., 2006; Dodla

and Bellamkonda, 2008). After it was cooled below 37 ºC, the gel was mixed with an equal

volume of microtubes loaded with chABC/ 1M trehalose, placed in a 96-well plate, and

gelled at 4 ºC. The final working concentration of the gel was 0.6% (w/v). After the mixture

gelled, 1X PBS was added above the mixture and incubated in a 37 ºC incubator for up to 2

weeks. During this incubation, the supernatant was collected and replaced with fresh 1X PBS

every other day. The collected supernatant was mixed with decorin, incubated at 37 ºC and

analyzed with DMMB assay as described in the section 3.2.3. The same measurement was

conducted with microtubes loaded with P‟ase (50 ng; same amount as chABC) and trehalose

as an enzyme control.

3.2.7 CHABC RELEASE PROFILES FROM LIPID MICROTUBES

The delivered amount can be determined by calculating the loading efficiency, which

is the ratio between the loaded volume of therapeutic agent and the entire internal volume of

the microtubes. After the release profile of the agent loaded microtubes is measured in vitro,

the daily delivered amount of agent in vivo can be predicted. Currently, there is no

54
commercially available kit for quantifying chABC levels or antibody to chABC, therefore,

the release profile of chABC was determined via SDS-PAGE and silver staining. The

samples of chABC released from the hydrogel-microtube mixture were prepared as described

in section 3.2.6, and the amount of chABC was determined via SDS-PAGE as described in

section 3.2.1 and silver staining as described in the section 3.2.2. Various different amounts

of chABC were prepared as a standard and run on SDS-PAGE at the same time with the

collected samples. Intensity measurements of the collected samples and chABC standards

were detected using a Microplate Reader (BIO-TEK Instruments, Inc.). The amount of the

released chABC was determined by the standard curve and the release profile was obtained.

3.2.8 NT-3 RELEASE PROFILES FROM LIPID MICROTUBES

NT-3 was loaded into lipid microtubes and the release profile of NT-3 was

determined by using methods similar to those previously published (Jain et al., 2006). An

NT-3 Sandwich ELISA Kit (Chemicon) was used to quantify the NT-3 release profile. NT-3

(PeproTech Inc.) containing microtubes were mixed with SeaPlaque® agarose gel, placed in

a 96-well plate, and cooled for gelation (30 ng per plate). The final working concentration of

the gel was 0.6 % (w/v). During incubation in a 37 ºC incubator for 2 weeks, the supernatant

was collected and replaced with fresh PBS every other day after sampling at day 1 and stored

at -80 ºC. The retrieved supernatants were pooled for the NT-3 ELISA and absorbance

measurements of the samples were taken at 450 nm using a Microplate Reader (BIO-TEK

Instruments, Inc.). The amount of the NT-3 was determined by the standard curve and the

release profile was obtained.

55
3.2.9 ANALYSIS OF TEMPERATURE DEPENDENT CONFORMATION OF CHABC

To investigate the potential of conformational changes contributing to the thermal

destabilization of chABC, we conducted circular dichroism (CD) studies with and without

trehalose stabilization. chABC was dissolved into 50 mM sodium phosphate (pH 7.4) and

placed in 10 mm light path length quartz cells. Scans collected measurements from between

195 and 300 nm with a 1nm bandwidth. For the denaturation curve, scans collected

measurements from between 5 and 85 ºC at a wavelength of 222 nm with a scan rate of 2.5

ºC/min. Four scans were averaged for each running condition. The transient temperatures

between fresh chABC in buffer and chABC in trehalose solution were compared.

3.3 RESULTS

3.3.1 FUNCTIONAL STABILITY OF CHABC AT BODY TEMPERATURE (37 ºC)

WITHOUT THERMAL STABILIZATION

We investigated the enzymatic activity of unstabilized versus trehalose-stabilized

chABC by evaluating its ability to digest the CSPG decorin followed by SDS-PAGE.

Decorin, our model CSPG, has a simple molecular structure consisting of one chondroitin or

dermatan sulfate GAG chain on its core protein. Intact decorin migrates as a larger molecular

weight broad smear on an SDS-PAGE gel (Fig. 3.3, Lane 3). A tighter and lower molecular

weight decorin band after digestion by chABC indicates preservation of chABC activity.

When the CS-GAG chain of decorin was digested by chABC, the core protein migrates at

~40-45 kDa (Fig. 3.3, Lane 1), and chABC alone migrates between 97 kDa and 116 kDa (Fig.

3.3, Lane 4). chABC pre-incubated for 24 hours at 37 ºC retained its ability to degrade

decorin (Fig. 3.3, Lane 2), but chABC lost the ability to completely degrade decorin GAG

56
chains after 1 week of pre-incubation at 37 º (Fig. 3.3, Lane 5). In contrast, following

incubation with 1 M trehalose at 37 ºC for one week, chABC retained its ability to degrade

decorin (Fig. 3.3, Lane 6).

1 2 3 4 5 6

Figure 3.3. SDS-PAGE assay for chABC enzymatic activity after 1

day pre-incubation and 1 week of pre-incubation at 37 ºC with and
without trehalose. Lane 1: fresh chABC + decorin, Lane 2: 1 day pre-
incubated chABC + decorin, Lane 3: intact decorin, Lane 4: chABC,
Lane 5: 1 week pre-incubated chABC + decorin, and Lane 6: 1 week
pre-incubated chABC with trehalose + decorin. Thermostability of
chABC was enhanced with trehalose and trehalose-chABC still
retained the ability to digest decorin after 1 week incubation at body
temperature.

3.3.2 TREHALOSE SIGNIFICANTLY ENHANCES THERMOSTABILITY OF CHABC

ENZYMATIC ACTIVITY

To improve the thermal stability of chABC, trehalose was introduced as a protein

stabilizer. To determine the proper concentration of trehalose, different concentrations of

trehalose, from 20 mM to 1 M, were evaluated for their ability to preserve chABC enzymatic

activity. Trehalose concentrations above 500 mM successfully preserve chABC enzyme

activity after 2 weeks of incubation at 37 ºC (Fig. 3.4).

57
 1 2 3 4 5 6

Figure 3.4. chABC enzymatic activity assay with different

concentrations of trehalose by SDS-PAGE. Each lane represents 1) 20
mM, 2) 50 mM, 3) 100 mM, 4) 250 mM, 5) 500 mM and 6) 1M of
trehalose-chABC with decorin after 2 weeks of pre-incubation at 37 ºC.

1 2 3 4 5 6 7

Figure 3.5. chABC enzymatic activity test after 4 weeks of pre-

incubation with and without trehalose at 37 ºC by SDS-PAGE. Lane 1:
4 weeks pre-incubated chABC with trehalose + decorin, Lane 2: 4
weeks pre-incubated chABC + decorin, Lane 3: fresh chABC +
decorin, Lane 4: decorin, Lane 5: fresh chABC + decorin, Lane 6:
fresh P‟ase + decorin, and Lane 7: fresh P‟ase (out of molecular
weight range from gel; ~28 kDa). The trehalose-thermostabilized
chABC still retained its activity to degrade CS-GAG of decorin after 4
weeks of pre-incubation at body temperature and P‟ase had no effect
on decorin CS-GAG.

58
 To investigate whether trehalose can preserve chABC activity at 37 ºC for longer

periods, 1M trehalose-chABC solutions were pre-incubated for 4 weeks at 37 ºC, and then

added to decorin for digestion. In figure 3.5, Lane 1 demonstrates that after 4 weeks of pre-

incubation with 1 M trehalose, chABC digested decorin, and confirms the ability of trehalose

to preserve chABC activity at 37 ºC. In comparison, after 4 weeks of incubation in PBS at 37

ºC without trehalose, chABC lost its enzymatic activity (Fig. 3.5, Lane 2). Decorin incubated

with fresh chABC (Fig. 3.5, Lane 3) and intact decorin (incubated for 4 hours at 37 ºC; Fig.

3.5, Lane 4) were run as controls at the same time. P‟ase was used as a control enzyme, and

when decorin was incubated with P‟ase at 37 ºC (Fig. 3.5, Lane 6), no decorin digestion was

observed as expected. P‟ase was not shown (P‟ase alone; Fig. 3.5, Lane 7) because molecular

weight of P‟ase (28 kDa) is out of range of this molecular standard weight.

3.3.3 TEMPERATURE STABILIZATION OF CHABC BY TREHALOSE IS DUE TO

CONFORMATIONAL STABILITY

Deactivation profiles of chABC enzymatic activity with and without trehalose over

time were evaluated with by the DMMB assay (Fig 3.6). At every time point except day 1,

there was a statistically significant difference between control and trehalose treated samples.

The enzymatic activity of chABC was maintained up to day 15 with trehalose. In comparison

however, without trehalose, chABC quickly lost its enzymatic activity after day 3 and was

completely inactive by day 5. Based on this deactivation study, a kinetic deactivation

constant kd was evaluated by assuming a two-state transition between native state (N), to

unfolded state (D):

ND

59
 dN/dt = − kbN; N= N0exp(− kbt)

where N0 represents initial N and the first-order kinetics was adapted (the dotted line in

figure 3.6). The deactivation rate constant of chABC was computed to be 0.27 [day-1;

R2=0.98]. The slope of the decline in chABC activity when it is thermostabilized by trehalose

is gentle and not precipitous and the kinetic deactivation constant of trehalose-chABC was

0.017 [day-1; R2=0.74].

To investigate whether increased thermal stability of chABC in the presence of

trehalose is due to conformational stabilization of the enzyme, conformational changes in

chABC as a function of temperature with and without trehalose were quantified by CD (Fig.

3.7). Ellipticity change measured by CD is directly proportional to the change in

concentration of native and unfolded forms; therefore CD has been applied to protein folding

study. The thermodynamics of protein conformational stability can be described by following

equations (Greenfield et al., 1999):

K = α / (1− α) (1)

∆G = nRTlnK; ∆G = ∆H − T∆S (2)

θobs = (a1-a2) α + a2 (3)

where K is the equilibrium constant of folding, α is the friction folded, ∆G is the free energy

of folding, R is the gas constant, T is temperature (Kelvin), ∆H is the enthalpy of folding and

∆S is the entropy of folding. θobs represents the observed ellipticity, a1 is fully folded

(maximum) θobs and a2 is fully native (minimum) θobs. Based on these equations, ∆H and the

midpoint transition temperature, Tm, were calculated by fitting the best curve to θobs.

60
 Figure 3.6. Kinetic analysis of chABC deactivation by DMMB assay. The
X axis represents days and the Y axis represents percentage of digested
decorin. Asterisks denote a significant difference from chABC in 1X PBS
(P < 0.05) and data represent mean ± SEM. The dotted line represents the
calculated deactivation curve of chABC in 1x PBS. Data are mean ± SEM.
chABC in 1X PBS (×) loses most activity within 5 days, and in contrast,
chABC in 1 M trehalose (Δ) retains its activity to degrade decorin CS-
GAG up to 15 days.

Initial values of ∆H, Tm, a1 and a2 were estimated, and calculated θ was fitted to the θobs by

curve fitting routine. The spectrum was measured at 222 nm and from 20 °C to 90 °C at the

rate of 2.5 °C/min and 1 min of delay. The Tm, ∆Hm and ∆Sm of chABC in 1 M trehalose

solution were 64.2 °C, 171 kJ∙mol-1 and 506 J K-1 mol-1 respectively, versus the Tm, ∆Hm and

∆Sm of chABC dissolved in sodium phosphate buffer were 56.2 °C, 176 kJ∙mol-1 and 533 J

61
K-1 mol-1. Therefore, the increment in the midpoint of transition, ΔTm, between the two

conditions is 8 °C, indicating that chABC‟s conformation is thermally more stable in the

presence of 1 M trehalose.

0.8

0.6
ΔA222nm

0.4

0.2

0
20 40 60 80 100
Temperature ( Cº)

Figure 3.7. The normalized thermal denaturation curves (fraction unfolded)

of chABC in pH 7.4 were measured by the changes of the absorbance at
222nm. The dashed line represents chABC in 50 mM sodium phosphate
buffer and the solid line represents chABC in 1M trehalose solution. Tm of
chABC in 1 M trehalose solution was 64.2 °C and the Tm of chABC
dissolved in buffer solution was 56.2 °C. Therefore, the increment in the
midpoint of transition, ΔTm, between the two conditions is 8 °C and can be
attributed to the presence of trehalose.

62
3.3.4 LIPID MICROTUBE ENCAPSULATED CHABC IS BIOLOGICALLY ACTIVE

FOR 2 WEEKS

To verify that self-assembled, hollow lipid microtubes (0.5 microns x 40 microns in

dimensions) can be loaded with chABC without compromising its enzymatic activity,

chABC released from fresh microtubes (without pre-incubation at 37 ºC) was tested with

SDS-PAGE (Fig. 3.8B) for its ability to digest decorin. As indicated by the digested decorin

band, microtube loading does not negatively impact chABC activity.

After confirming that lipid microtubes can be used for chABC delivery, post-release

activity of chABC or P‟ase from microtubes was plotted (Fig. 3.8A) as a function of time,

and their relative enzymatic activity was measured by DMMB assay (Melrose and Ghosh,

1988; de Jong et al., 1989). 100% of digested decorin on the Y-axis represents 1X PBS with

no decorin. This release profile showed that, when combined with trehalose, chABC released

from microtubes retained its enzymatic activity up to 15 days in vitro. As expected, the

combination of the control enzyme P‟ase with trehalose did not digest of decorin.

63
 (A)

(B)

Figure 3.8. Enzymatic activity of post-released chABC from lipid microtubes.

(A) Quantification of the amount of CS-GAGs that remained after digestion of
decorin with the released enzymes by DMMB assay. chABC (Δ) and P‟ase (×)
with trehalose/microtubes. The Y-axis represents percentage of digested
decorin and the X-axis represents time. All data points are significantly
different (P < 0.05; mean ± SEM). (B) SDS-PAGE assay of chABC released
from trehalose/microtube + decorin.

64
3.3.5. RELEASE PROFILE OF MICROTUBE ENCAPSULATED NT-3 CONFIRMS A

SUSTAINED DELIVERY FOR 2 WEEK

To characterize the release profile of NT-3, NT-3 released from microtubes was

tested with NT-3 sandwich ELISA kit to measure released amount (Fig. 3.9).

50
Accumulated NT-3 (ng)

40

30

20

10

0
0 2 4 6 8 10 12 14
Days

Figure 3.9. Release profile of NT-3 from the hydrogel-microtube delivery

scaffold.

3.4 DISCUSSION

The purpose of this study was to develop an approach that allows the application of

chABC as a therapeutic agent in vivo. chABC is one of the more promising therapeutic

interventions for SCI, however, the thermal instability of chABC compromises its potential

for in vivo application (Tester et al., 2007). In this study, we demonstrate that the enzymatic

activity of chABC could be maintained at body temperature over time and that

thermostabilized chABC delivered by a hydrogel-lipid microtube scaffold system over two

65
weeks remains enzymatically active in vitro. In this chapter, it is shown that this hydrogel-

microtube delivery scaffold has a potential to provide an alternative delivery method to

chronically implanted, invasive pumps, which is minimally invasive and effectively delivers

chABC in vivo.

After 24 hour incubation at 37 ºC, the enzymatic activity of chABC, which is used in

this study, was still preserved. However after 1 week of incubation at 37 ºC, a broaden

decorin band appeared (Fig. 3.3, Lane 5), indicating that enzymatic activity was weakened

leading to incomplete digestion of CS-GAGs on decorin even after sufficient reaction time.

At body temperature chABC quickly loses most of its enzymatic activity within 5 days (the

DMMB assay; Fig. 3.6; ×-dashed line) and the retained enzymatic activity is not enough to

completely digest CS-GAG chains, though partial digestion is still possible (seen as a

broaden band in SDS-PAGE assay; Fig. 3.3). chABC typically lost most of its enzymatic

function within one week at body temperature, however the rate of enzymatic activity loss

and the amount of enzymatic activity retained depend on several factors. The SDS-PAGE

assay showed different retained activities of chABC from different lots. Other studies also

showed that enzymatic thermostability of chABC depends on lot and time (Tester et al.,

2007). Large differences exist between lots, since function can be lost anywhere from 1 day

to 2 weeks, so all experiments in this study were performed with chABC from the same lot.

Biodegradable polymer nanoparticles also could be considered as a carrier for

sustained delivery of chABC (Cohen et al., 1991; Genta et al., 2001; Kim and Burgess, 2002;

Singh et al., 2009). Before using the lipid microtube, poly(lactic-co-glycolic acid) (PLGA)

nanoparticles were examined as drug carriers for chABC in a preliminary. The double

emulsion method was used to make PLGA nanoparticles, and is simple, takes a relatively

66
short time and is easy to store in powder form at -80 ºC after lyophilizing comparing to the

fabrication procedure of lipid microtubes. After fabricating powder form of PLGA

nanoparticle encapsulating chABC, the enzymatic activity of chABC released from the

PLGA particles was characterized with the same assessment described in the section 3.2.1.

However the mark band (Fig. 3.3, Lane 1) presenting core protein due to GAG chain

digestion by chABC was not observed and a broad band of decorin, similar to the one in

Lane 5 of figure 3.3, was seen in the gel. chABC lost its enzymatic activity after the

fabrication procedure of PLGA nanoparticles. It is possible that proteins undergo physical

denaturation and chemical degradation during fabrication. The acidification of the internal

PLGA microenvironment and the water-in-oil-in-water (w/o/w) encapsulation procedure are

considered major protein stresses inducing inactivation and aggregation (van de Weert et al.,

2000; Perez-Rodriguez et al., 2003; Estey et al., 2006). During the double emulsion

procedure, chABC is exposed to organic solvents, dichloromethane, and the pH of inside

particles is predominantly below pH 5.8 (detection limit in this paper, Li and Schwendeman,

2005).

Our laboratory has previously reported the use of lipid microtubes for the slow

delivery of a variety of proteins (Meilander et al., 2001), DNA (Meilander et al., 2003; Yu

and Bellamkonda, 2003; Meilander-Lin et al., 2005) and neurotrophic factors (Yu and

Bellamkonda, 2003; Jain et al., 2006) in vivo. Relative to other delivery systems such as

polyester based PLGA nanoparticles, lipid microtubes have the advantage that there is no

exposure to heat or organic solvents that might cause denaturation or degradation, during the

fabrication process. As is evident from figure 3.8B, release from lipid microtubes does not

adversely affect the enzymatic activity of chABC. In addition to that, lipid microtubes are

67
injectable either by themselves, or when embedded in thermo-reversible hydrogels as

reported in Jain et al. (2006), Dodla et al. (2008) and Chvatal et al. (2008).

Here, the lipid microtube-gel delivery system provides sustained delivery of

thermostabilized chABC over a period of 2 weeks, with the microtubes enabling slow release

while the hydrogel localizes the tubes to the lesion site. We have developed a mathematical

model (Meilander et al., 2001), to predict the release profile of compounds with a range of

molecular weights such as myoglobin (17.8 kDa), albumin (66.4 kDa) and thyroglobulin

(660 kDa). These studies suggest that an initial burst release of protein from day 1 to day 3 is

followed by a slow and continuous release, leading to a cumulative percentage of released

agents of 80-100% by day 14. Further, microtubes show a stable and continuous release

profile with delivery of BDNF (Brain derived neurotrophic factor) (Jain et al., 2006), and

NT-3 (neurotrophin-3) (Dodla and Bellamkonda, 2008).As the molecular weight of chABC

(120 kDa) is within the protein molecular weight range previously tested, most of the chABC

loaded into microtubes is likely released by day 14.

In this study, we chose to stabilize chABC by adding trehalose since it is known as an

exceptional stabilizer and widely used with proteins (Carninci et al., 1998; Kreilgaard et al.,

1998). The molecular mechanism of trehalose-mediated thermostabilization is still poorly

understood (Hedoux et al., 2009). There are several hypotheses outlining the mechanisms of

protein stabilization by trehalose – water replacement (Crowe et al., 1984), preferential

hydration (Timasheff, 2002), vitrification of solutions (Green et al., 1989) and the influence

of trehalose on the water tetrahedral hydrogen bond network (Branca et al., 1999). Previous

works have shown that surface tension of trehalose solutions increases linearly with increase

in trehalose concentration (Kita et al., 1994), and there is a strong correlation between

68
surface tension and increase in Tm (Kaushik and Bhat, 2003). Studies have shown that the

dynamic fluctuation of polar side chains at the solvent-protein interface was reduced in the

presence of trehalose (Hedoux et al., 2009). The increased surface tension or the limited

exposure of hydrophobic groups to the water molecules leads to preferential hydration,

resulting in the stabilization of the tertiary structure of protein (Timasheff, 2002). In this

study, the Δ Tm of chABC (8 °C) with 1M trehalose addition is similar in range to its effects

on other enzymes such as RNase A (5.5 °C) and lysozyme (8.4 °C) (Kaushik and Bhat, 2003),

where increased conformational thermostabilization (increase of Tm) induces an increase in

enzymatic activity. These enzymes also show an increase of ΔTm when concentration of

trehalose increases, and it describes the observation that only high concentration (500 mM

and 1 M) of trehalose preserved the enzymatic activity of chABC in this study. Therefore,

trehalose‟s ability to thermally stabilize chABC is consistent with its effects on other

enzymes.

3.5 CONCLUSIONS

In this study, improvement of thermostability of chABC enzymatic activity was

attained by adding trehalose up to 4 weeks. We demonstrated that thermostabilized chABC

can be slowly delivered by the lipid microtube-hydrogel delivery system and the released

chABC retains its enzymatically activity over two weeks in vitro. Therefore, these

achievements of thermostabilization and development of the sustained delivery system

provide an alternative and novel application for chABC treatment after SCI.

69
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73
 CHAPTER 4

DELIVERY OF THERMOSTABILIZED CHABC BY IMPLANTING HYDROGEL-

MICROTUBE SCAFFOLDS AFTER SPINAL CORD INJURY AND EXAMINATION

OF CELLULAR AND MOLECULAR RESPONSES: SHORT TERM STUDY

(Partially as published with R.J. McKeon and R.V. Bellamkonda, Proceedings of the

National Academy of Science, 2009 )

Chondroitin sulfate proteoglycans (CSPGs) are upregulated and accumulate around

the lesion site after spinal cord injury (SCI), and are major inhibitors of regeneration. To

overcome CSPG-mediated inhibition, modification or digestion of CSPGs with

chondroitinase ABC (chABC) has been explored. chABC digests glycosaminoglycan (GAG)

chains on CSPGs and enhances sprouting and regeneration when delivered to the lesion site.

However, chABC has a crucial limitation; it is thermally unstable at body temperature (37ºC)

and its enzymatic activity is significantly attenuated within 3 days. Therefore, multiple or

continuous infusions with an indwelling catheter are necessary to maintain enzymatic

functionality for periods of 2 weeks or longer and such devices are invasive and clinically

problematic. To overcome these limitations, we enhanced the thermal stability of chABC by

adding a protein stabilizer, trehalose, and demonstrated that chABC maintained its enzymatic

activity for 4 weeks at 37 ºC in vitro. Also, we developed a delivery scaffold for chABC in

vivo application in the temporally and spatially controlled manner.

In this chapter, a dorsal over hemisection injury was made at thoracic vertebrae T10

in adult rats, and thermally stabilized chABC was delivered to the lesion site by a minimally

74
invasive hydrogel-microtube scaffold carrying chABC-loaded lipid microtubules at the lesion

site immediately following injury. To determine the effectiveness of topical delivery of

thermostabilized chABC, animal groups treated with single injection or hydrogel scaffold

implantation of chABC, chABC/trehalose and P‟ase were included as controls. Two weeks

after surgery, the enzymatic functionality of released chABC in vivo was examined by CS-56,

WFA and 3B3 immunostaining.

The results demonstrated that thermally stabilized chABC was successfully delivered

slowly and locally without a catheter/mini-pump and the released chABC effectively

digested CSPGs in vivo and significant differences of CSPG digestion were observed in 3B3-

IR and CS-56-IR between groups. We suggest that chABC could be combined with other

therapies or agents, such as neurotrophic factors to treat SCI.

4.1 INTRODUCTION

After injury, a series of cellular and molecular events occur, and results in the

formation of astro-glial scar tissue consisting of interwoven reactive astrocytes and

chondroitin sulfate proteoglycan (CSPGs). The CSPG-astroglial scar provides a non-

permissive environment for axon regeneration. Studies have shown that CSPGs are

upregulated after injury in the CNS and neurite regrowth is inhibited by CSPGs (McKeon et

al., 1995; Stichel et al., 1995; Lemons et al., 1999; Jones et al., 2002). It has been shown that

growth cone stall or change growth orientation at the CSPG-rich region in vitro and in vivo.

Therefore, it is important to remove or attenuate the inhibitory nature of CSPGs by

modifying CSPGs and to encourage axons to regrow through the inhibitory regions around

the lesion site. Therefore, chABC has been used to treat SCI, based on the concept that

75
removing or attenuating inhibitory activity of CSPGs could promote axonal regeneration.

After promising results for axonal outgrowth were reported in vitro (Snow and Letourneau,

1992; Zuo et al., 1998) and in vivo (Yick et al., 2000; Moon et al., 2001; Bradbury et al.,

2002; Yick et al., 2003; Chau et al., 2004; Caggiano et al., 2005; Barritt et al., 2006; Houle et

al., 2006), it has received a lot of attention as a potential therapy for spinal cord injury.

chABC shows the best enzymatic activity at 37 ºC and the optimum pH is near 8.0.

At physiological conditions, body temperature 37 ºC and 7.4 pH, chABC should be suited for

in vivo usage. However, as described in the previous chapters, there are crucial limitations

when delivering chABC for SCI treatment, such as thermal stability of chABC and

difficulties with spatio-temporal control. In general, CSPGs are upregulated and deposited

around the lesion site for at least 2 weeks after the initial injury, and the CSPG-rich matrix

surrounding the lesion site persists for up to 8 weeks following injury (Jones et al., 2003).

Therefore, for chABC to degrade CSPG associated glycosaminoglycans (GAGs), a “fresh”

supply would need to be delivered intrathecally for at least two weeks. Currently this is

achieved via intrathecal injection, with the infusion frequency varying from days to weeks,

and for time periods ranging from 2 to 6 weeks to compensate the rapid loss of enzymatic

activity of chABC. A single administration of chABC containing gelfoam on lesion site of

T11 hemisection promotes Clarke‟s nucleus neurons to regrow beyond the injury (Yick et al.,

2000; Yick et al., 2003). Multiple or continuous infusions were also administrated, using an

osmotic mini-pump (Chau et al., 2004; Houle et al., 2006; Huang et al., 2006) and catheters

with externalized cannulas (Moon et al., 2001; Bradbury et al., 2002; Caggiano et al., 2005;

Barritt et al., 2006).

76
 Diffusion of chABC into deep regions of the cord, however, is limited when delivered

intrathecally due to overflow into the intrathecal space, thereby diluting the enzyme rapidly

and necessitating concentrated infusions (from 2 U/ml to 1000 U/ml) to compensate. With

the exception of catheters with externalized cnnulas, the efficiency of CSPG digestion is

dependent on the enzyme‟s stability at body temperature. Therefore, there is a compelling

need to develop clinically viable methods for the spatially and temporally controlled delivery

of „fresh‟ chABC, preferably in a manner confining it to the lesion site. We demonstrated an

alternative method using trehalose-thermostabilized chABC and a hydrogel and lipid

microtube based minimally invasive system to deliver chABC in a temporally and spatially

controlled manner in vivo in chapter 3.

Therefore, the study in chapter 4 was designed to investigate chABC‟s activity in vivo

at the lesion site after SCI when delivered via our lipid microtube-hydrogel system and

compare the CSPG digestion efficiency to a single injection of chABC. In this short term

(two week) animal study, we locally delivered chABC to the lesion site by a topical delivery

model and the dorsal over-hemisection injury was used. Thermostabilization of chABC and

development of sustained delivery scaffold were achieved previously in the chapter 3. Two

weeks after surgery, the animals were sacrificed, and the functionality of chABC released

from the gel scaffold was examined. CS-56 and 3B3 immunostaining were used to examine

the level of CSPGs and CSPG digestion by chABC‟s activity, and WFA was used to

visualize perineuronal nets, which encapsulate synapses throughout the nervous system with

CSPGs.

77
4.2 MATERIALS AND METHODS

4.2.1 FABRICATION OF MICROTUBE-HYDROGEL SCAFFOLDS

The fabrication procedure of the lipid microtube was previously published (Meilander

et al., 2001; Meilander et al., 2003) and described in the Chapter 3, section 3.2.4. The

hydrogel-microtube delivery scaffolds were fabricated by loading chABC/ 1M trehalose

solution into the microtubes, and mixed with an equal amount of 2% (w/v) SeaPrep® agarose

gel (Cambrex) in 1X PBS for 1% of final gel concentration. Before mixing SeaPrep agarose

gel with the microtubes, the gel solution was filtered for sterilization and cooled to below 37

ºC to avoid deactivation of chABC or P‟ase due to high temperature. After complete gelation

at 4 ºC, each gel scaffold contained 10mU of chABC prior to being implanted in SCI animals,

as described below, section 4.2.2.

4.2.2 TOPICAL DELIVERY OF HYDROGEL-MICROTUBE SCAFFOLDS IN A

DORSAL OVER HEMISECTION INJURY MODEL

Adult male Spraque-Dawley rats (Charles River Laboratory) weighing between

235~260 g were anesthetized with 2% of isoflurane and maintained during surgery with 0.5%

of isoflurane. The surgical area was shaved and the skin and muscles were opened to expose

the thoracic vertebrae T9-T11. A single laminectomy was performed on T10 to expose the

spinal cord by removing the bone with a micro-rongeur and the dura mater was excised.

Using micro-scissors marked at 1.5 mm depth from the tip, a controlled dorsal hemisection

injury (2 mm of width x 1.5 mm of depth) was made by cutting the dorsal columns at T10.

Sustained topical delivery of thermostabilized chABC (MTC; n=8) was achieved using

methods previously published (Chvatal et al., 2008). Briefly, the SeaPrep agarose hydrogel

78
embedded with lipid microtubes loaded with either chABC/1M trehalose or chABC alone

was placed on top of the lesion site as shown schematically in figure 4.1. To stabilize the

hydrogel embedded microtubes in place, a stiffer 0.7% of SeaKem agarose hydrogel solution

was added on top. This denser gel was quickly gelled in situ by a stream of cooled air (Fig.

4.2) as described in a previous study (Jain et al., 2006). After ensuring complete gelation, the

muscles were sutured together and the skin was closed with wound clips. Figure 4.3

demonstrates the topical delivery model applied into the spinal cord lesion site.

Table 1 describes experimental and control groups. As controls, 10 mU of chABC in

5 µL of 1X PBS (SC; n=6) or 1 M trehalose/1X PBS (STC; n=6) was injected as „single

injection‟ conditions that have local, but not sustained delivery. 100 ng of P‟ase (Sigma) was

delivered by single injection (STP; n=6) or hydrogel-microtube scaffold (MTP; n=6) with

1M trehalose as a negative enzymatic delivery control for chABC specificity. Sham (Sham;

n=4) and trehalose loaded hydrogel scaffold conditions (MT; n=6) were also conducted. To

avoid differences in the tissue reaction caused by absence of SeaKem agarose gel, after a

single injection of agents, a pre-made SeaKem block was placed on top of the lesion site.

After two weeks, the animals were anesthetized with a ketamine-xylazine-acepromazine

cocktail (1:0.17:0.37 ml/kg) and transcardially perfused with 4% paraformaldehyde.

79
Figure 4.1. Schematic of spinal cord injury model and delivery of enzyme to
lesion site. The 1 % SeaPrep agarose gel-microtube-scaffold is implanted on
top of the lesion and covered with stiffer 0.7% SeaKem agarose gel to keep
the scaffold in place.

Figure 4.2. Schematic of gel cooling system. Tubing runs from the nitrogen
gas tank to a Styrofoam box containing 100% ethanol and dry ice, and runs
through an aluminum rod inside a tube containing dry ice to keep the
nitrogen gas cool. The cooled nitrogen gas was applied over the stiffer
agarose gel solution for in situ gelation, which covers the top of the gel-
microtube delivery scaffold. Figure from Jain et al., 2006.

80
 R

A C B

C D
C

Figure 4.3. This figure demonstrates the topical delivery model applied into
the spinal cord lesion site. A) Exposed intact spinal cord at T10 level after a
single laminectomy. B) Dorsal over hemisection injury made with a single
cut to the spinal cord column. C) Gel scaffold, embedded with chABC loaded
microtubes, implanted on top of the lesion site. D) The implanted hydrogel-
microtube delivery scaffold was covered with stiffer agarose gel to stabilize
its location on top of the lesion site. (R = rostral, C = caudal)

81
 Table 1. Experimental design of 2 week study with notation
Notation of
Components # of rats
groups
Agarose gel scaffold embedded with
MTC microtubes loaded with chABC and 8
trehalose
Agarose gel scaffold embedded with
MT 6
microtubes loaded with trehalose
STC Single injection of chABC with trehalose 8
SC Single injection of chABC 6
Agarose gel scaffold embedded with
MTP microtubes loaded with penicillinase and 6
trehalose
Single injection of penicillinase with
STP 6
trehalose
NoT Injury and no treatment 6
Conducted the same procedure except
Sham 4
injury with other groups

M: lipid microtubes, S: single injection, T: trehalose, C: chABC and P: penicillinase

4.2.3 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY OF SPINAL

CORDS

At two weeks post-surgery, the animals were perfused transcardially with 1X PBS

followed by 4% paraformaldehyde in 1X PBS. The T9-T11 spinal cord was removed, post-

fixed in 4% paraformaldehyde and placed in 30% sucrose at 4 ºC until it sank to the bottom.

Tissues were frozen in OCT and sagittal sections were cut with a cryostat (HM 560MV

Cryostat) at 14 µm thickness. All sections were mounted in serial order onto charged

microscope slides.

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 Sections were washed with 0.5% Triton X-100 PBS for 10 min and incubated with 4%

of goat serum/0.5% Triton X-100 PBS (blocking solution) for 1 hour. The sections were

incubated with primary antibodies diluted in blocking solution at 4 ºC overnight. The

following primary antibodies were applied: an antibody to the stub protein after chABC

digestion (1:150, mouse IgM, clone 3-B-3; Seikagaku America) which recognizes

unsaturated, C6-sulfated glycosaminoglycan stubs (Baker et al., 1991); chondroitin sulfate

proteoglycan (1:250, mouse IgM, clone CS-56; Sigma) to identify CSPGs; glial fibrillary

acidic protein (GFAP) (1:600, polyclonal rabbit IgG; Chemicon) for astrocytes; and Wisteria

floribunda agglutinin (WFA) (5 µg/ml, biotin conjugate; Sigma) for perineuronal nets. After

primary antibody incubation, sections were washed three times with 0.5% Triton X-100 PBS

and incubated with the appropriate secondary antibodies conjugated to fluorophores for 1

hour at room temperature. The following secondary antibodies were used: goat anti-rabbit

IgG(H+L) Alexa Fluor 488 (1:200 dilution in 0.5% Triton X-100 PBS; Invitrogen) for GFAP;

goat anti-mouse IgM Alexa Fluor 594 (1:200) for 3B3 and CS-56; and streptavidin Alexa

Flour 488 for WFA. Sections treated with secondary antibody but without primary antibody

were used as staining controls. The sections were washed three times with 1X PBS and DAPI

was applied to some of the sections for 15 min at room temperature to label the cell nuclei.

The sections were washed twice with 1X PBS and then coverslipped using Fluoromount-G

(Southern Biotechnology Associates, Inc.).

To examine the effectiveness and enzymatic functionality of chABC delivered by

hydrogel-microtube delivery system and single injection treatments, triple staining was done

with 3B3, GFAP and WFA, and adjacent sections were double stained with CS-56 and GFAP.

GFAP labeling was used to delineate the border of the astroglial scar lining the lesion site

83
(Fig. 4.4A). Images were taken on the Zeiss Axioskop 2 Plus microscope (Zeiss, Thornwood,

NY) with an Olympus Microfire digital camera.

4.2.4 QUANTITATIVE ANALYSIS OF CSPG DIGESTION AND ASTROCYTE

RESPONSE

Quantification of CSPG digestion

To quantify the 3B3-immunoreactivity (IR) and CS-56-IR after chABC delivery

around the lesion site, four or five micrographs were taken at 20x magnification along the

lesion site as figure 4.4A. Relative to the lesion site, two caudal and two rostral images as

well as one image underneath the lesion were used. At least four animals were chosen from

each animal group and relative fluorescent intensity was measured using ImagePro software

package (MediaCybernetics). The gross fluorescent intensity was measured for the region

and a mean value was obtained and averaged for each animal. Background intensity was

measured on each section and deducted from the measured 3B3-IR and CS-56-IR intensity.

Quantification of GFAP

To quantify the astrocyte responses, the spinal cord tissues were immunostained and

imaged at 20x magnification along the lesion site with the same exposure time and conditions.

The images were analyzed with a custom-built image analysis program (MATLAB;

Mathworks) (Jain et al., 2006). This program generates line profiles radial to the defined

interface and records the intensity of the fluorescent signal along the line profile, thus

quantifying the relative value of intensity as a function of distance from the lesion interface

(x=0) into the spinal cord (Fig. 4.4B). The number of line profiles (e.g. 30 lines), the color

(one of RGB) to be read and the lesion interface can be selected by the user. With each run,

84
the relative intensity is averaged over all the line profiles. Four scans were performed for

each staining image and the results were averaged again over all four scans. Four sections

were chosen from each animal, four animals were chosen from each animal group and at

least five images were captured. At least 80 images (five images X four spinal cord sections

X four animals) were used and the relative intensity as a function of distance was averaged

over 2400 (30 lines X 80 images) line profiles per experiment group. After this analysis, the

relative intensity was compared between groups for different distances from the interface: 0-

100, 100-300 and 300-500 µm.

A B

Figure 4.4. Micrographs of the GFAP immunostained tissue and method of

image analysis with a custom developed MATLAB program. A) 4x
immunostained image. The solid line represents the lesion boundary defined
by GFAP immunoreactivity and the boxed areas denote regions selected for
quantification. Scale bar is 500 µm. B) One of the boxes in figure A is
expanded at 20x magnification to show analysis of fluorescent intensity using
line profiles. The X axis represents distance from the lesion interface in pixel
unit. The lines were generated from the lesion interface into the tissue,
averaged and displayed as a function of distance from the interface to between
experimental groups. Scale bar is 100 µm.

85
4.2.5 STATISTICAL ANALYSIS

Minitab software was used to determine the statistical differences existing between

experimental conditions using analysis of variance (ANOVA). A p-value <0.05 was

considered statistically different.

All animal protocols were approved by the Institutional Animal Care & Use

Committee at Georgia Institute of Technology. Animal welfare, including pre-care, surgeries,

pain management, post-care and monitoring was supervised by the Animal Research

Committee of the Georgia Institute of Technology.

4.3 RESULTS

4.3.1 SUSTAINED DELIVERY OF ENCAPSULATED CHABC DIGESTS CSPGS

EFFECTIVELY IN VIVO

The effectiveness and enzymatic activity of trehalose stabilized chABC when

delivered in vivo after SIC using our lipid microtube-embedded hydrogel delivery system at

the lesion site (Fig. 4.1) was examined by CS-56 and 3B3 immunostaining. The 3B3

antibody recognizes unsaturated, C6-sulfated glycosaminoglycan stubs (Baker et al., 1991)

and therefore, can be used as a marker for CSPGs digested by chABC. The CS-56 antibody

was used to identify intact CSPG deposition. GFAP immunoreactivity (GFAP-IR) was used

to identify reactive astrocytes and in combination with either 3B3 or CS-56, to define the

lesion boundary. Two weeks after hemisection SCI, tissue sections from the MTC (hydrogel-

microtube delivery scaffold loaded with 1M trehalose/chABC; See Table 1 for notation of

groups) treated animals showed significantly strong 3B3-IR near the lesion site (Fig. 4.5E)

86
while CS-56-IR in adjacent tissue sections was diminished (Fig. 4.5D). CS-56-IR was strong

along the lesion interface in most of the tissue sections, however intensity of CS-56-IR was

significantly reduced overall for the region near the lesion with sustained chABC delivery. In

the no-treatment (NoT) group, the reverse IR was observed with strong CS-56 intensity and

no 3B3-IR (Fig. 4.5A and B). Similar patterns were observed in the rest of the other control

groups except the MTC group.

To determine if chABC delivery affected the integrity of PNNs in the vicinity of the

lesion site, PNNs were labeled with Wisteria floribunda agglutinin (WFA) cytochemistry in

the same tissue sections that were stained with 3B3. WFA is a lectin specific for N-

aceylgalactosamine and results in visualizing net-like structures of PNNs. PNNs were

observed near the lesion site in single injection-trehalose-chABC, no treatment (Fig. 4.5C)

and other conditions except the MTC treated condition where significantly less WFA staining

was observed (Fig. 4.5F). Immunostaining of WFA, 3B3, and CS-56 showed that WFA-

PNNs were present in conditions when CS-56-IR intensity was strong and 3B3-IR was weak

or absent around the lesion site. In the MTC treated group, the 3B3 positive region where

CSPGs were degraded by chABC had no WFA-PNNs and low CS-56-IR. Therefore

sustained delivery of chABC resulted in significantly diminished PNNs close to the lesion

interface implicating the potential for greater axonal sprouting.

87
 C

D E F

Figure 4.5. Immunohistological analysis of CSPG digestion in vivo (A-F).

Images were taken right next to the lesion boundary of no treatment (A, B
and C) and MTC treatment (hydrogel-microtube delivery scaffold loaded
with chABC/ 1M trehalose; D, E, and F) animals. CS-56-IR for intact
CSPGs (A and D). 3B3-IR for digested CSPGs (B and E). WFA staining for
perineuronal nets (C and F). The intensity of CS-56-IR and WFA is
inversely proportional to 3B3-IR. Arrows in (C) indicate WFA-PNNs. Scale
bar is 100µm.

88
4.3.2 QUANTIFICATION OF 3B3, CS-56 AND GFAP IMMUNOSTAINING

Measurements of fluorescent pixel intensity were as used to quantify 3B3, CS-56 and

GFAP immunostaining (Fig. 4.6) using an ImagePro software and a custom-built MATLAB

routine. 3B3-IR is significantly higher in the MTC group as compared to all other groups

(Fig.4.6A, p<0.05). The average CS-56-IR is significantly lower in the MTC group than

others and there is a statistical difference for CS-56-IR between MTC and no-treatment

(NoT); MTC and STP (single injection-trehalose-P‟ase) (Fig. 4.6B, p<0.05).

GFAP-IR positive astrocytes were used to define the astro-glial scar region and the

boundary of the lesion site (Fig. 4.4A). The fluorescent pixel intensity was quantified with a

line profile image analysis program developed in MATLAB (Fig. 4.4B; Jain et al., 2006); the

program measures intensity changes from the lesion interface into the spinal cord radially.

The intensity of GFAP-IR decreased from the lesion site into the cord and the number of

reactive astrocytes also decreased, and correlated positively with CS-56-IR intensity.

Analyzed intensity was divided into three bins, 0-100, 100-300 and 300-500 µm away from

the lesion interface. The GFAP-IR was lower in the MTC treated group than other groups,

and there is a significant difference between the MTC treated group and other groups; NoT,

STP and MT in 0-100 µm and 100-300 µm (Fig. 4.7, p<0.05). This data confirm that our

sustained delivery system using lipid microtubes embedded in agarose hydrogel does not

negatively impact the astrogliotic response.

89
 *

Immunostaining intensity

A 3B3

*
Immunostaining intensity

B CS-56

Figure 4.6. Quantitative image analysis of 3B3-IR and CS-56-IR fluorescent intensity.
The X axis represents each experimental condition treated for animal groups. The Y
axis represents the relative fluorescent intensity of IR. The relative fluorescent
intensity was measured alone the lesion boundary, and mean value was obtained and
averaged for each animal. (A) 3B3-IR quantitative analysis. Asterisk denotes
significant increase of 3B3-IR in MTC treatment compared to all other treatments
(p<0.05). (B) CS-56-IR quantitative analysis. Asterisks denote significant decrease of
CS-56-IR in MTC treatment when compared to NoT and STP treatments (p<0.05).
No significant differences were observed among other treatments. The data represent
the mean ± SEM.

90
 200
* *
noT STP
180
MT SC
GFAP intensity

STC MTP
160
MTC
140

120

100

80

60
0 100 200 300 400 500

Distance from the lesion site (µm)

Figure 4.7. Quantitative image analysis of GFAP-IR fluorescent intensity by line

profile. The X axis represents distance from the lesion interface into the spinal cord in
µm and 0 represents the lesion interface delineate the border of the astroglial scar
lining the lesion site. The Y axis represents the relative fluorescent intensity of
GFAP-IR. Overall the intensity of GFAP-IR decreased from the lesion interface into
the cord as a function of distance. The GFAP-IR was lower in the MTC treated group
than other control groups. The IR intensity was analyzed by dividing into three bins,
0-100, 100-300 and 300-500. Asterisks denote a significant difference between the
MTC treated group and other control groups; NoT, STP and MT in 0-100 µm and
100-300 µm (P <0.05). The data represent the mean.

91
4.4 DISCUSSION

The thermal instability of chABC represented a significant impediment to

regeneration after SCI, as it limited drug delivery approaches and forced the use of infusion

pumps or catheters for delivery. In this study, we demonstrate that the enzymatic activity of

chABC could be maintained at 37 ºC by adding trehalose and that thermostabilized chABC

delivered by a lipid microtube-hydrogel scaffold system remains enzymatically active for at

least two weeks in vivo. This study therefore demonstrates that a single treatment with of the

hydrogel-microtube system provides an effective and creative alternative to chronically

implanted, invasive pumps/catheters that are typically used to deliver chABC in vivo.

Given that astro-glial scar deposition spans a period of 2 to 4 weeks post-injury,

clinical application of chABC might require enzymatic activity over a period of weeks. Most

studies to date have delivered chABC via multiple intrathecal injections to achieve prolonged

action. The time required to reach peak deposition of CSPG at the lesion site is dependent on

the type of CSPG (Jones et al., 2003). NG2 is the predominant CSPG expressed after SCI

and reaches peak deposition between 1 and 2 weeks after injury, and the expression levels of

neurocan, brevican, and versican are elevated after SCI, peak at 2 weeks and are maintained

for 4 weeks or more. The production of several CSPGs and proteoglycan species is

differently affected by degree and type of spinal cord injury. The main deposition pattern is

that CSPG molecules are significantly elevated within 24 hours, slowly increase until they

peak and then decrease, and overall CSPG-rich matrix persists up to 2 months. We chose a

time point of 14 days after surgery to analyze the effectiveness of thermostabilized chABC

and our delivery system because overall CSPG expression levels are highest at 14 ~ 18 days

and decrease by 49 days (Iseda et al., 2008). Delivery of active agents during this period

92
would be expected to affect inflammation and cellular responses, modulate CSPGs

deposition around lesion site, and promote axonal sprouting and outgrowth.

The sustained and local delivery of chABC was achieved over a 2 week time period

using a previously characterized gel scaffold system in our laboratory. It has been

demonstrated that agarose hydrogel-lipid microtube-based delivery acts as a sustained

delivery system in vitro with various proteins and DNA (Meilander et al., 2001; Meilander et

al., 2003). An in situ technique is used to quickly cool and set the liquid gel, and this gel

cooling system (Fig. 4.2) allows the gel to mold into the shape of the injury site. A BDNF-

loaded hydrogel-lipid microtube delivery scaffold was used to fill the lesion cavity on the

dorsal column of the spinal cord (Jain et al., 2006), and methylprednisolone-loaded

biodegradable PLGA nanoparticle-hydrogel was topically delivered to the contusion -injured

spinal cord as described in figure 4.1 (Chvatal et al., 2008; Kim et al., 2009). This gel-

microtube system also can be combined with a conduit to provide a bridge with neurotrophic

factors for peripheral nerve regeneration (Dodla and Bellamkonda, 2008). The previous

studies and this study demonstrate that our gel-microtube based delivery system does not

aggravate inflammatory responses compared to control groups. By implanting the stabilized

chABC sustained delivery scaffold on the top of the lesion site, we avoid a second surgery

that could cause additional injury and stress to the animal. We also avoid a chronically

inserted catheter/mini-pump that could induce inflammatory mass and increase infection risk

(Penn, 2003; Peng and Massicotte, 2004).

In the MTC treated group, 3B3-IR is significantly higher compared to all other groups.

As a control, to compare the effect of sustained delivery versus single administration, a dose

of thermostabilized chABC (STC) equal to the total amount of chABC released from the

93
lipid-microtubes over 14 days was injected at the lesion site. This single injection had no

effect as determined by a lack of 3B3 staining. This data indicates that indeed sustained

release of chABC is critical either because chABC diffuses away and loses its enzymatic

activity quickly after the single injection, or because CSPGs continue to be produced over

time. In the MTC treated group, 3B3-IR is significantly higher compared to all other groups.

It is interesting that in the STC group, small bright spots of 3B3-IR were observed around the

lesion site (gray matter) possibly due to chABC digesting some CSPGs before being washed

out. Once CSPGs are digested, the turn-over is relatively slow. PNNs were also degraded by

the digestion of CSPGs following microtube-mediated release of chABC. In comparison, a

single injection of chABC was not sufficient to break-down PNNs. In the MTC treated

spinal cord, PNNs were not observed immediately around the lesion site, but PNNs were

present approximately 1 mm away from the lesion site suggesting that thermostabilized

chABC activity was localized to the region in the immediate vicinity of the lesion.

Permissive environment

Another consequence of chABC treatment is the effects of the disaccharidic

degradation products of CSPG generated by chABC digestion. The disaccharidic by-product

protects neurons against inflammation-induced neurodegeneration by down-regulating T cell

motility and decreasing cytokine secretion such as interferon-γ and tumor necrosis factor-α

(Rolls et al., 2006). The by-product modulates intracellular signaling pathways such as PKCα

and PYK2, and induces neuronal outgrowth and protects neurons against neuronal toxicity

(Rolls et al., 2004). In this study, GFAP-IR quantification demonstrates that a) there is no

elevation of inflammation response in gel scaffold treated groups, and b) GFAP-IR was

significantly lower in the MTC treated group than others (NoT, STP and MT) near the lesion

94
area (Fig. 4.7). Therefore, it is possible that chABC treatment generated disaccharidic

degradation products act as neuroprotective agents and trigger neuronal protective and

survival signaling pathways.

4.5 CONCLUSIONS

In conclusion, this study clearly demonstrates that sustained local delivery of

thermostabilized chABC digests CSPGs after SCI without aggravating the secondary injury

response. Combinational therapy of chABC and neurotrophic factors via our delivery system

could provide a synergic effect on axonal regrowth and functional recovery after SCI. This

approach obviates the need for invasive, pump mediated delivery of chABC, elegantly

enhances the thermostability and resultant efficacy of chABC in vivo, and overcomes an

important technical hurdle for the in vivo application of chABC therapy after SCI.

95
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 CHAPTER 5

DELIVERY OF THERMOSTABILIZED CHABC AND NT-3 AND EVALUATION

OF AXONAL REGENERATION AND FUNCTIONAL RECOVERY AFTER SPINAL

CORD INJURY: LONG TERM STUDY

(Partially as published with R.J. McKeon and R.V. Bellamkonda, Proceedings of the

National Academy of Science, 2009)

CSPGs are one major class of axon growth inhibitors which are upregulated after SCI

and contribute to regenerative failure. Chondroitinase ABC (chABC) digests CS-GAG chains

on CSPGs and can thereby overcome CSPG mediated inhibition and promote axonal

regeneration when delivered at the site of injury. However, chABC loses its enzymatic

activity rapidly at 37 ºC, necessitating the use of repeated injections or local infusions with a

mini-pump or catheter for days to weeks. Maintaining these infusion systems is invasive,

infection-prone and clinically problematic. To overcome this limitation, we thermostabilized

chABC and developed a system for its sustained local delivery in vivo, obviating the need for

chronically implanted catheters and pumps in chapter 3. When stabilized with trehalose,

chABC remained active at 37 ºC in vitro for up to 4 weeks. A lipid microtube-agarose

hydrogel system was used for controlled release of chABC over 2 weeks at the lesion site

following a SCI in chapter 4. The enzymatic functionality of released chABC and the cellular

and molecular responses were examined by immunostaining with 3B3, CS-56, GFAP and

WFA two weeks after injury. The results demonstrate significant differences in CSPG

99
digestion between groups treated with the chABC-loaded hydrogel microtube delivery

system and controls.

In this chapter, a long term study (45 days) was conducted to examine axonal

regeneration and functional recovery after chABC treatment and combination treatment with

a neurotrophic factor, NT-3. The resultant impact of the initial CSPG digestion during the

first two weeks was still present at 6 weeks following SCI when delivered by a hydrogel-

microtube delivery system. Axonal growth and functional recovery following the sustained

local release of thermostabilized chABC versus a single treatment of unstabilized chABC

demonstrated significant differences in CSPG digestion. Additionally, animals treated with

stabilized chABC in combination with sustained NT-3 delivery showed significant

improvement in locomotor function, enhanced growth of CTB-positive sensory axons, and

sprouting of 5-HT serotonergic fibers at the lesion site. We suggest that this significant

improvement of chABC thermostability facilitates development of a minimally invasive

method for sustained, local delivery of chABC that is potentially effective in overcoming

CSPG-mediated regenerative failure. Combination therapy of thermostabilized chABC and

neurotrophic factors enhances axonal regrowth, sprouting and functional recovery after SCI.

5.1 INTRODUCTION

After injury to the central nervous system, the lesioned axons fail to re-grow or make

functional connections (Schwab and Bartholdi, 1996). While the cellular and molecular

mechanisms of axon growth failure are active areas of research, a breakthrough clinical

therapy has yet to be developed. Injuries to the CNS induce a series of inflammatory events

around the lesion site, typically resulting in permanent functional loss.

100
 There are many factors contributing to the failure of spontaneous regeneration; a lack

of sufficient neurotrophic support (Widenfalk et al., 2001), a lack of intrinsic regeneration

capacity in CNS as compared to PNS microenvironment (Neumann and Woolf, 1999),

inhibitory molecules such as myelin-associated proteins (Filbin, 2003; Schwab, 2004;

Schwab et al., 2006) and glial scar-associated CSPGs (McKeon et al., 1995; Silver and Miller,

2004), which are upregulated after injuries to the CNS and contribute to regenerative failure.

Strategies can be developed for each inhibitory factor to overcome the lack of repair capacity

after spinal cord injury. In chapter 3 and 4, we focused on developing a chABC treatment to

attenuate CSPG-mediated failure of axonal regeneration and provide permissive substrates

prepared by chABC digestion for axonal outgrowth.

In this chapter a combination strategy was developed to provide a neurotrophic factor

for chemo-attracting axons to the lesion site. Combination therapies with chABC have been

previously investigated. Schwann cell-seeded guidance channels were implanted into

hemisected adult rats after chABC treatment (Chau et al., 2004) and an autologous peripheral

nervous system bridge with cellular matrix modified by chABC was grafted onto a

hemisection lesion (Houle et al., 2006). Synergistic effects were observed on axonal

sprouting into the dorsal column nuclei after combination treatment with chABC and NT-3

(Massey et al., 2008). Retinal fiber sprouting with combination treatment with chABC and

BDNF after denervation of the superior colliculus has been also observed (Tropea et al.,

2003). To encourage axonal outgrowth, we chose to use NT-3 with chABC. Removal of an

unfavorable environment (CSPG) by chABC and presentation of the supporting neurotrophic

factor NT-3 was expected to be a promising combination for SCI.

101
 A longer 45 day study was designed to examine the functional consequences of

CSPG digestion by chABC and a combination therapy with the neurotrophic factor NT-3 via

implantation of a hydrogel-lipid microtube delivery system at the lesion site. We

demonstrated that the enzymatic activity of chABC can be thermostabilized at 37 ºC due to

the ability of trehalose to confer conformational stability, and that thermostabilized chABC

delivered by a lipid microtube-hydrogel scaffold system is enzymatically active in situ for at

least 2 weeks as described in Chapters 3 and 4. Here, a combination treatment of

thermostabilized chABC and NT-3 was used to overcome the CSPG-rich astroglial barrier to

axonal regeneration and encourage axonal outgrowth. Locomotor behavioral improvements

with combination delivery of chABC and NT3 were observed, and correlated well to

immunohistological evidence of axonal sprouting at the lesion site. Analysis of 5-HT sensory

fibers and CTB-labeled fibers demonstrated that chABC treatment can induce plasticity of

injured projections within the spinal cord and offers a possible mechanism of chABC-

induced functional recovery. This study therefore demonstrated that a single administration

of the hydrogel-microtube delivery system provides an effective alternative to chronically

implanted, invasive catheters or mini-pumps typically used to deliver chABC after SCI. The

combination therapy of chABC and NT-3 with our delivery method offers a potent approach

for clinical application after SCI.

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5.2 MATERIALS AND METHODS

5.2.1 TOPICAL DELIVERY OF HYDROGEL-MICROTUBE SCAFFOLDS IN A

DORSAL OVER HEMISECTION MODEL

Table 5.1 describes conditions and notations of experimental and control groups in

the 45 day in vivo study. In the long term study, MTC (hydrogel-microtube delivery scaffold

loaded with 1M trehalose/chABC; n=7), MTP (hydrogel-microtube delivery scaffold loaded

with 1M trehalose/P‟ase; n=6) and sham (n=4) treatment conditions were used, the other

conditions used in the short term study were excluded, and three new treatments were added.

In the short term study, a single injection of chABC did not show effective digestion of

CSPGs, and there was no significant difference from no treatment animals in the 2 week

study (in chapter 4, section 4.3.2). Therefore, single injection controls (SC – single injection

of chABC, STC – single injection of 1M trehalose/chABC, STP – single injection of 1M

trehalose/P‟ase) were excluded.

MTN (hydrogel-microtube-1M trehalose/NT-3), MTCN (hydrogel-microtube-1M

trehalose/chABC and 1M trehalose/NT-3) and GC (hydrogel mixed with an equal amount of

1M trehalose/chABC to the MTC treatment condition) treatment conditions were added for

the long term study. As a control to probe the sustained release enabled by lipid microtubes

embedded in the hydrogel, 1 % of SeaPrep agarose gel mixed with 10 mU of chABC/1M

trehalose was implanted on the top of the lesion site (n=6; GC); the other conditions included

lipid microtubes loaded with NT-3 (n=7; MTN; 100 ng per rat) or combination microtubes

loaded with chABC and NT-3 (n=8; MTCN) to investigate effect of combination treatment

of chABC and neurotrophic factor.

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 The surgical procedure followed to induce injury and the delivery methods of

hydrogel-microtubes delivery scaffold were the same as for the 2-week short term study

described in the chapter 4, section 4.2.2. Briefly, adult male Sprague-Dawley rats (Charles

River) received a dorsal over-hemisection injury at the T-10 vertebral level. Sustained topical

delivery was achieved using methods previously reported (Chvatal et al., 2008) and shown in

the figure 4.1 and figure 4.3.

5.2.2 RETROGRADE NEUROAL TRACER INJECTION INTO THE SCIATIC NERVE

Six weeks post-injury and 3 days before sacrificing, cholera toxin B subunit (CTB;

sigma) was injected into the right sciatic nerve for retrograde axonal tracing. Rats were

anesthetized using isoflurane gas. The thigh region on the right leg was shaved, and 2 cm of

incision was made through the skin and muscles to expose the sciatic nerve. 5 µl of 1% CTB

was slowly injected into the nerve with a 34 gauge needle (NanoFil). The muscles were

sutured and the skin closed using wound clips. 3 days after injecting the CTB, the animals

were the animals were anesthetized with a ketamine-xylazine-acepromazine cocktail

(1:0.17:0.37 ml/kg) and perfused transcardially with PBS followed by a mixture of 4%

paraformaldehyde in PBS to facilitate fixation.

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 Table 2. Experimental design of 45 days study with notation
Notation of # of
Components
groups rats
Agarose gel scaffold embedded with
MTC 7
microtubes loaded with chABC/trehalose
Agarose gel scaffold embedded with
MTN 7
microtubes loaded with NT-3
Agarose gel scaffold embedded with
MTCN microtubes loaded with chABC/trehalose 8
and NT-3
GC Agarose gel mixed with chABC/trehalose 6
Agarose gel scaffold embedded with
MTP microtubes loaded with 6
penicillinase/trehalose
Conducted the same procedure except
Sham 4
injury with other groups

M: lipid microtubes, S: single injection, T: trehalose, C: chABC and P: penicillinase

5.2.3 BEHAVIORAL ANALYSIS

Locomotion and thermal pain sensitivity were assessed to determine functional

improvement in the 45 day long term study. The investigators were blinded with regard to

animal groups throughout these tests. CatWalk (Noldus), a video-based analysis system, was

used to assess locomotor deficits in voluntarily walking. Each rat voluntarily walked across

the walkway three times every week after injury. After acquiring raw data, paw prints were

labeled as right forepaw (RF), left forepaw (LF), right hindpaw (RH) and left hindpaw (LH)

and measurements of locomotion were provided by the software. Stride length, paw print

pattern and base support were chosen to examine behavior as a function of time after injury

105
for all experimental conditions. Stride length was defined as a distance between consecutive

steps with the same limb. The distance in mm between the two hind paws was defined as the

base of support and this distance is measured perpendicular to the direction of walking. The

white boxes in figure 5.1 show abnormal hindpaw print patterns and the number of abnormal

prints was counted for three step cycles.

Figure 5.1. CatWalk raw data in false color mode. White boxes represent
abnormal hindpaw print patterns. The dotted lines represent the stride length
defined as a distance between consecutive steps with the same limb and the
solid line represents the base of support defined as a distance between the two
hind paws. (dark red – left hindpaw, light red – left forepaw, dark green – right
hindpaw and light green – right forepaw)

Thermal sensitivity assessment by a dynamic plantar test (Ugo Basile) was used,

identical to the method of Hargreaves et al (1988). The device measured the time taken to

elicit a flexion reflex. Each hind-paw was tested three times every week and averaged. The

animals were placed on a glass floor under which a mobile radiant infra-red heat source was

positioned at the animal‟s hind paw. When the animal withdrew its paw, the source power

turned off, triggered by an automatic sensor.

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5.2.4 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY

At 45 days post-surgery, the animals were perfused transcardially with PBS followed

by 4% paraformaldehyde in PBS. The T9-T11 spinal cord was retrieved, post-fixed in 4%

paraformaldehyde and placed in 30% sucrose at 4 ºC. Tissues were frozen and sagittal

sections were cut with a cryostat (HM 560MV Cryostat) at 14 µm thickness. All sections

were mounted in serial order onto charged microscope slides.

Sections were washed with 0.5% Triton X-100 PBS for 10 min and incubated with 4%

of goat serum/0.5% Triton X-100 PBS (blocking solution) for 1 hour. The sections were

incubated with primary antibodies diluted in the blocking solution at 4 ºC overnight. The

following primary antibodies were applied against: CS-56 (1:250, mouse IgM; Sigma) to

identify CSPGs; glial fibrillary acidic protein (GFAP) (1:600, polyclonal rabbit IgG;

Chemicon) for astrocytes; anti-CTB (1:600; abcam); and serotonin (1:150, 5-HT, monoclonal

mouse IgG1; Abcam) for serotonergic neurons. After primary antibody incubation, sections

were washed three times with 0.5% Triton X-100 PBS and incubated with the appropriate

secondary antibodies conjugated to fluorophores for 1 hour at room temperature. The

following secondary antibodies were used: goat anti-rabbit IgG(H+L) Alexa Fluor 594

(1:200 dilution in 0.5% Triton X-100 PBS; Invitrogen) for GFAP; goat anti-mouse IgM

Alexa Fluor 488 (1:200) for anti-CTB, 5-HT serotonin and CS-56. Sections treated with

secondary antibody but without primary antibody were used as staining controls. The

sections were washed three times with PBS and coverslipped using Fluoromount-G.

To evaluate the axonal sprouting, double staining was conducted with CTB and

GFAP or 5-HT and GFAP. Double staining of 3B3 and GFAP was also conducted to

examine the level of CSPGs. GFAP labeling was used to define the border of the astroglial

107
scar lining the lesion site (Fig. 4.4A). Images were taken on the Zeiss Axioskop 2 Plus

microscope with an Olympus Microfire digital camera.

5.2.5 QUANTITATIVE ANALYSIS OF AXONAL SPROUTING

A montage of each tissue section stained for CTB labeled fibers was obtained at 10x

magnification using Neurolucida software (MicroBrightField Bioscience). The 0 indicates

the caudal lesion interface and defined by GFAP immunoreactivity (IR). The relative

intensity of CTB labeled fibers were measured using ImagePro software (MediaCybernetics)

at 1 and 0.5 mm caudal to the start of lesion, at the start of the lesion, and 0.5 and 1 mm

rostral to the lesion. The intensity of CTB labeled fibers 1 mm caudal to the caudal edge of

the lesion was assumed to represent the total CTB fibers approaching the lesion site and the

percentage of CTB fibers stopped within the defined regions was determined by measuring

the relative intensity. Background intensity was measured on each section and deducted from

the measured CTB intensity.

To quantify 5-HT serotonin immunofluorescence, micrographs were taken at 20x

magnification from two defined regions, ventral gray matter caudal and rostral to the lesion

site, and relative fluorescent intensity was measured by ImagePro software package. The

gross fluorescent intensity was measured for the region and mean value was obtained.

Background intensity was measured on each section and deducted from the measured 5-HT

IR intensity.

108
5.2.6 STATISTICAL ANALYSIS

Minitab software was used to determine the statistical differences existing between

experimental conditions using analysis of variance (ANOVA). A p-value <0.05 was

considered as statistically different.

All animal protocols were approved by the Institutional Animal Care & Use

Committee at Georgia Institute of Technology. Animal welfare, including pre-care, surgeries,

pain management, post-care and monitoring was supervised by the Animal Research

Committee of the Georgia Institute of Technology.

5.3 RESULTS

5.3.1 LEVELS OF CSPG DEPOSITION AFTER SUSTAINED DELIVERY OF CHABC

AT 6 WEEKS

To examine whether the resultant impact of the initial 2 weeks of CSPG digestion

persists in vivo after 6 weeks, CS-56 immunostaining for intact CSPGs was conducted. Tissue

sections from the MTCN treated animals showed that significantly less CS-56 expression

compared to MTP treated controls (Fig. 5.2).

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 Figure 5.2. Micrograph of CS-56-IR around the lesion site at 6 weeks. (A)
MTCN and (B) MTP. CSPG-IR is significantly less in the MTCN treated
animals compared to MTP treated controls. Scale bar is 100 µm.

5.3.2 SUSTAINED DELIVERY OF CHABC AND NT-3 IMPROVES LOCOMOTOR

FUNCTION

Functional recovery was assessed by using the CatWalkTM and thermal plantar tests

throughout the 45 day period. A significant difference in locomotor function was observed

six weeks post-injury, but not earlier, and no improvement was observed with the thermal

pain threshold test in any animal groups throughout the six week testing period. All the

animal groups showed improved locomotor function throughout the testing period. At day 7

and day 14, all animals showed abnormal walking patterns, slow crossing, and abnormal paw

prints, reflecting the SCI-mediated dysfunction. Four weeks post surgery, most animals

showed a normal step sequence and the time necessary to cross the walkway decreased. A

number of abnormal hind paw prints also decreased and stride length increased due to

recovery from injury. However the differences between animals were not observed by 4

weeks.

110
 Figure 5.3 showed CatWalk raw data of sham (A), MTCN (B) and MTP (C) treated

animals on the walkway in false color mode at 6 weeks. Dark red represents left hindpaw;

light red – left forepaw, dark green – right hindpaw and light green – right forepaw. As seen

on figure 5.3, hindpaws step on the print of the consecutively previous forepaw. Animals

with higher degree injuries generally fail to walk with a normal step sequence and step on the

consecutively previous forepaw print. In this study, we used a relatively moderate injury

(dorsal over hemisection), therefore, most animals showed normal step patterns. Six weeks

post-SCI surgery, there were significant differences in the stride length between treatment

conditions (Fig. 5.4). Average stride lengths (mean ± SEM) were 183.6 ± 6.91 mm for

MTCN, 172.89 ± 20.8 mm for MTC, 157 ± 18.25 mm for MTN, 152.91 ± 13.13 mm for GC

and 154.07 ± 10.11 mm for MTP at 6 weeks (See Table 2 for notation of groups). Significant

differences were observed between MTCN and GC (p = 0.019), and MTCN and MTP (p =

0.035).

At 6 weeks, the MTCN, MTC and MTN treated animals had more normal foot prints

(Fig. 5.3B) compared to MTP treated animals (white boxes in Fig. 5.3C). The sham animal

had normal foot print and the MTP animal had abnormal hindpaw prints (white box in Fig.

5.3C.). The number of abnormal hindpaw prints was counted per three step cycles and the

averages were 0.7 ± 0.3 for MTCN, 1.7 ± 0.6 for MTC, 1.8 ± 0.5 for MTN, 2.1 ± 1.3 for GC

and 3.3 ± 1.3 for MTP. The mean number of abnormal print of the single treated animals,

MTC and MTN, is less than that of animals treated by GC and MTP and the combination

treatment MTCN group, on average had even fewer abnormal foot prints. However while

these were strong trends, the differences were not significant between animal groups.

111
 The distance in mm between the two hind paws was defined as the base of support

and this distance is measured perpendicular to the direction of walking. Animals tend to

show a larger base of support after SCI. At 6 weeks, the averages were 31.4 ± 4.8 mm for

MTCN, 30.7 ± 4.4 mm for MTN, 39.8 ± 8.9 mm for GC and 38.5 ± 4.5 mm for MTP, and

there was no statistical difference between animal groups.

112
Figure 5.3. CatWalk raw data in false color mode, (A) Sham, (B) MTCN
and (C) MTP treated animals on the walkway at 6 weeks. All animal
groups show a normal step sequence. The white box in the figure C
represents an abnormal hindpaw print. Red represents left paw prints and
green represents right paw prints (dark red – left hindpaw, light red – left
forepaw, dark green – right hindpaw and light green – right forepaw).

113
 210
† *
† Sham
190 MTC
MTN
Stride length (mm)

† MTCN
170
MTP
GC
150

130

110
2 week 4 week 6 week

Figure 5.4. Stride length analysis for locomotion functional recovery. Data are
mean ± SEM and asterisk denotes statistical significance between MTCN and
MTP, and between MTCN and GC (P <0.05). Sham showed significant
differences († < 0.05) compared to all other conditions throughout the testing
period, except for MTCN at 4 and 6 weeks.

114
5.3.3 SUSTAINED DELIVERY OF CHABC AND NT-3 PROMOTES SPROUTING

5.3.3.1 AXONAL SPROUTING AROUND THE LESION AREA: CTB-LABELED

FIBERS

To examine anatomical regeneration in the dorsal column, CTB was injected into the

sciatic nerve and resulted in labeling of the primary apparent within the spinal cord. The

location of labeled fibers was examined; the MTP and GC treated animals had retracted

CTB-labeled fibers in the glial scar area and few fibers approached the lesion site (Fig. 5.5A).

However in the MTC, MTN and MTCN treated animals, more CTB-labeled fibers crossed

the glial scar area and approached the lesion boundary (Fig. 5.5B). CTB-labeled fibers grew

around cavities along the lesion interface (Fig. 5.5C and D; 0 to 0.5 mm interval in C and 0.5

to 1 mm interval in D) and a few fibers were observed after 0.5 mm rostral to caudal edge of

the lesion in chABC and NT-3 treated animals. However no fibers entered the lesion cavity

in any animal groups. Significantly more fibers crossed 0 and 0.5 mm rostral to the caudal

edge of the lesion in the MTCN (p=0.03 and p=0.045; p=0.049 and p=0.045, respectively)

animals compared to GC and MTP treated animals (Fig. 5.6). At one mm rostral to the caudal

edge of the lesion start, a few fibers were observed in the MTC, MTN and MTCN treated

animals, however most were not strong enough to be detected by the relative intensity

measuring method described in section 5.2.5.

115
Figure 5.5. Micrographs of CTB labeled fibers (green) at the lesion site at
6 weeks. The dashed lines represent the lesion interface. (A) MTP and (B)
MTCN at 10 x magnification. Scale bar is 500 µm (C) An expanded figure
from the white box 0 to 0.5 mm interval in figure A, and (D) an expanded
figure from the white box in 0.5 to 1 mm interval figure B at 20 x
magnification. Arrows represent CTB labeled fibers. Scale bar is 100 µm.

116
 100
MTC
% of crossed CTB-labeled fibers

80 MTN
MTCN
60 MTP
GC
40 *

20
*

0
-0.5 0 0.5 1
Distance from the caudal edge of the lesion (mm)

Figure 5.6. Quantification of CTB+ axon growth. The Y axis represents

the percentage of crossed axons at the distance to the lesion interface and
the X axis represents distance to the lesion (mm). The data represent the
mean ± SEM. Asterisks denote a significant difference compared with
MTCN (P<0.05).

117
5.3.3.2 AXONAL SPROUTING AROUND THE LESION AREA: 5-HT-

IMMUNOREACTIVE FIBERS

5-HR-IR fibers in the ventral gray matter were measured to quantify the percentage of

the lesion site. More 5-HT-IR fibers were observed in the gray matter of MTC, MTN and

MTCN treated animals. The serotonergic fibers were observed in the gray mater, primarily in

the ventral horn and lamina X, and more fibers were located rostral versus caudal to the

lesion (Fig. 5.7A). The spinal cords tissues treated with chABC, NT-3 and combination of

chABC and NT-3 had higher fluorescent intensity and the 5-HT-IR fibers extended closer to

the lesion site than MTP and GC treated controls. Quantification confirmed that MTCN

treatment showed significant sprouting of serotonergic fibers caudal to the lesion versus all

other conditions (p = 0.008 for MTC, p = 0.024 for MTN, p = 0.001 for GC and p = 0.001 for

MTP); and rostral to the lesion than MTN (p = 0.015), GC (p = 0.002) and MTP (p = 0.001)

treatment (Fig. 5.8).

118
 caudal rostral

B C

Figure 5.7. Immunohistological analysis of 5-HT-IR fibers. (A)

Micrograph of 5-HT at 4× magnification in the MTCN treated animal
tissue. The boxed areas in A denote regions selected for quantification and
the solid white line represents the lesion interface. More fibers were
located rostral versus caudal to the lesion. Scale bar is 500 µm. (B, C)
Expanded figures from the white box in figure A. Serotonergic
innervations rostral to the lesion in MTP (B) and MTCN (C) animals at
20× magnification. MTCN treated animal has higher fluorescent intensity
and also had extended closer to the lesion site than MTP treated animal.
Scare bar is 100 µm.

119
 caudal rostral
18
*
16 MTC
14
% of 5-HT-IR fibers

MTN
12
MTCN
10
* MTP
8
GC
6
4
2
0
location to the lesion

Figure 5.8. Quantitative analysis of 5-HT-IR intensity for the stained

spinal cord. Quantification demonstrated that caudal to the lesion, MTCN
showed significantly (* p <0.05) increased 5-HT-IR compared to all other
treatments, and rostral to the lesion, MTCN showed significantly (* p
<0.05) increased 5-HT-IR compared to all other treatments except for
MTC. Data are mean ± SEM.

120
5.4 DISCUSSION

A six week long term study was performed to determine whether chABC or NT-3

delivered individually by using the hydrogel-microtube delivery system would encourage

axonal sprouting/regeneration and improve functional recovery after SCI in the rodent model.

Also, combination treatment of chABC and NT-3 was tested via our delivery system to

evaluate whether the combination therapy can provide synergic effect on axonal regrowth

and functional recovery. CSPG deposition, axonal sprouting and behavioral improvement

were assessed to determine efficacy of the hydrogel-microtube delivery system and

combination therapy. CS-56 immunostaining shows that the resultant impact of the initial

CSPG digestion is still present at 6 weeks. Locomotor behavior improved with combinational

chABC-NT3 delivery, and correlated well with increased axonal sprouting at the lesion site.

This study demonstrates that a single treatment of thermostabilized chABC via the hydrogel-

microtube delivery system provides an effective alternative to chronically implanted,

invasive pumps typically used to deliver chABC in vivo. In addition, the combination

treatment with NT-3 significantly enhanced functional recovery.

Once CSPGs are digested, the turn-over is relatively slow (Bruckner et al., 1998). In

the short term study, significantly decreased CSPG-IR was observed in the animals treated

with the chABC loaded hydrogel-microtube system. CS-56 immunostaining was performed

to examine whether CSPG digestion during the first two week still present. CS-56 staining at

6 weeks (Fig. 5.2) shows that little CS-56 expression persists compared to untreated controls.

This suggests that thermostabilized chABC digestion was effective early, at the peak of

CSPG production and its turn-over is slow (more likely), or that chABC activity via

microtubes is sustained for longer than 2 weeks (unlikely, but possible). In either case, the

121
chABC digestion via microtubes remains effective over a period of 6 weeks as is evident

from little CS-56 positive tissue at the 6 week time point. CSPG levels remain low up to 6

weeks post-injury, thereby facilitating sprouting and regeneration. Another study reported

similar results, showing that evidence of chABC digestion remained for at least 7 weeks in

the spinal cord (Galtrey et al., 2007).

The results demonstrated that a single treatment of chABC and NT-3 enhanced

sensory axonal sprouting, and the combination treatment enhanced more axonal sprouting

around the lesion site. The CTB labeled fibers mainly represent ascending sensory pathways

in the dorsal columns of the spinal cord. No CTB-labeled fibers entered the lesion cavity in

any treatment condition, however a significantly higher percentage of axons were approached

the lesion boundary and grew around the lesion site in the animals treated with single or

combination delivery of chABC and NT-3 compared to other groups. While some sensory

functional recovery was expected because of the result of CTB-fiber quantification, no

improvement was observed using the thermal pain threshold test (hindpaw withdrawal

latency to heat stimuli) in any of the animal groups during the 45 day test period. There are

two possibilities to explain the results; first, after the dorsal over hemisection cutting there

may be possibly spared fibers around the dorsal lateral fasciculus that help retain sensitivity

in all groups independent of lesion. Second, despite the significant primary afferent

sprouting in the treated animals, the sprouting was predominantly located in the dorsal white

matter and no fiber entered into the lesion site in any animal groups. Therefore, the axonal

sprouting was not enough to transmit stimuli to the dorsal horn where primary afferent

terminals are located, explaining why no differences exist between experimental groups. Our

observation is consistent with other studies. Despite robust primary afferent sprouting in

122
animals treated with chABC, no improvement of noxious thermal sensation (Barritt et al.,

2006) was observed, and transgenic mice expressed chABC under the murine gfap promoter

showed nearly complete digestion of CSPGs and robust axonal growth into the glial scar, but

no axon crossing into the lesion or motor functional recovery (Cafferty et al., 2007).

Enhanced locomotor functions were observed in the MTC and MTCN animal groups

and there were significant improvements in MTCN treated animals in terms of stride length,

but not base support or number of abnormal hindpaw patterns. Stride length is an important

parameter of locomotor efficacy as it impacts speed of locomotion (Hamers et al., 2001). The

gains in other locomotor functions were probably obscured by our choice of the relatively

mild hemisection injury model. Quantification of 5-HT serotonergic fiber sprouting

demonstrated significantly higher sprouting in MTCN group relative to all other groups. As

microtube-mediated delivery resulted in significant sprouting, with combinatorial delivery

eliciting even more significant sprouting, it is possible that the sprouted local serotonergic

spinal circuits facilitated observed locomotor improvements in stride length. This increase in

sprouting is likely due to chABC mediated digestion of CSPG-rich PNNs that surround

synapses (Vitellaro-Zuccarello et al., 1998). One of consequence of the chABC treatment is

the digestion of CSPG-rich perineuronal nets (PNNs) (Massey et al., 2006; Galtrey et al.,

2007; Massey et al., 2008). There is evidence that this structure regulates neuronal plasticity

(Pizzorusso et al., 2002), protects encapsulated neurons (Bruckner et al., 1999) and supports

ion homeostasis (Bruckner et al., 1993). This observation is consistent with other reports that

PNN digestion by chABC leads to increased plasticity and functional improvement due to

reinnervation and sprouting; PNN breakdown in the cuneate nucleus promoted collateral

sprouting/plasticity, resulting in axonal sprouting in the forelimb afferents after SCI (Massey

123
et al., 2006) and increasing plasticity in the spinal cord improved peripheral nerve

regeneration (Galtrey et al., 2007). CSPG removal by chABC delivery in the adult visual

cortex to treat monocular deprivation increased ocular dominance plasticity by significant

recovery of dendritic spine density (Pizzorusso et al., 2006). In this study we observed that

the PNN breakdown around the lesion area after chABC treatment (Fig. 4.5); motor neurons

and many interneurons are surrounded by PNNs. It may possibly have caused the increased

sprouting of serotonergic fibers, thereby increasing plasticity of spinal locomotor circuits and

resulting in functional locomotor recovery.

The de-stabilized PNNs by chABC digestion would promote anatomical plasticity by

increasing the density of newly-grown processes in the adult CNS ECM, and it could lead

functional plasticity. chABC-induced plasticity, such as collateral sprouting (Massey et al.,

2006) or aberrant sprouting (Barritt et al., 2006), is one major mechanism of chABC

treatment. Therefore, possible negative effects are aberrant sprouting and neuropathic pain

due to the aberrant plasticity (Woolf and Salter, 2000) or autonomic dysreflexia due to

increased plasticity (Weaver et al., 2006). These concerns have been raised previously.

Studies reported that no evidence of hyperalgesia was observed after chABC injection in to

the spinal cord (Pizzorusso et al., 2006), and no increase of connectivity of nociceptive

neurons and development of mechanical allodynia or thermal hyperalgesia was observed

even after aberrant sensory fiber sprouting (Barritt et al., 2006). Therefore, it is very

important to balance between detrimental sprouting and beneficial sprouting, and the

molecular mechanism involved in chABC-mediated improvement of plasticity needs to be

proven to develop the optimized chABC treatment after SCI.

124
 In the short term study, there appears to be significant impact of chABC digestion at 2

weeks, though evidence of functional recovery for in vivo studies was not seen until at least 6

weeks. With regard to the time lag between peak CSPG deposition and first evidence of

functional recovery, axonal regrowth and sprouting can occur after CSPG digestion – hence

the lag in time between CSPG digestion and functional recovery. After injury, severed axons

retract from the lesion site, therefore, functional recovery requires axonal regrowth and

sprouting to reconnect/regain the functional pathway. Once our chABC treatment removes

inhibitory molecules (CSPGs), the CSPG/astroglial-inhibitory region becomes permissive for

axonal outgrowth, and axons can then regrow and sprout through the region. After SCI,

animals are typically allowed 6 weeks or longer to assess axonal regeneration and functional

recovery.

Our data is consistent with other studies where successful digestion of CSPGs does

not automatically lead to improved behavioral outcomes (Barritt et al., 2006; Cafferty et al.,

2007). Transgenic mice having a gfap promoter to express chABC demonstrated almost

complete CSPG digestion at the reactive astrocyte-region. Significant sensory axon sprouting

was observed, however it was not sufficient to improve motor function (Cafferty et al., 2007).

It suggests that providing a permissive environment by removing inhibitory components is

not enough to induce significant functional improvement; combination therapy needs to

provide to favorable environment for sufficient axon regeneration. When chABC delivery is

combined with NT-3 lentivirus delivery, dramatically increased axonal extension was

observed compared to single delivery of chABC or NT-3 (Massey et al., 2008). In this study,

combination treatment of chABC and NT-3 resulted in significantly increased axonal

sprouting as compared to a single treatment of each.

125
 To apply this method as a clinical therapy, the practicality of chABC treatment

needs to be considered. In this study, the chABC activity was limited to 1 mm away

from lesion site and it is not clear whether this area is large enough to be clinically useful.

Because chABC is a relatively large molecule, diffusion through neural tissue and

extracellular space is limited. However, we believe that compared to intrathecal delivery

of chABC by a mini-pump, chABC delivered via the hydrogel-microtube system diffuses

deeper into the tissue (up to 1mm). This is because mini-pump delivery affects a larger

region, diluting the chABC, whereas our delivery system enables local delivery into the

tissue. As CSPG deposition tapers off exponentially from the lesion border, the local

delivery of chABC is relevant and effective, and sufficient diffusion (again as evident

from our 6 week CS-56 staining) occurs to effectively digest the CSPGs deposited near

the lesion site. In humans, there might be a need for greater diffusion distances as the

CSPG deposition zone may be deeper, but whether or not chABC diffusion into injured

human cord will be a limitation or not remains to be determined. The minimum clinically

relevant chABC concentration required to elicit behavioral recovery also remains to be

determined.

5.5 CONCLUSIONS

In conclusion, we demonstrate that sustained local delivery of thermostabilized

chABC digests CSPGs after SCI without aggravating secondary injury response.

Combination therapy of chABC and NT-3 facilitated by our sustained delivery system

enhanced axonal sprouting and functional recovery after SCI. This approach elegantly

126
obviates the need for invasive, indwelling catheter/pump-mediated delivery of chABC,

enables combinatorial therapy with neurotrophic factors, and represents a promising

approach to implementing chABC therapy after SCI.

127
5.6 REFERENCES

Barritt AW, Davies M, Marchand F, Hartley R, Grist J, Yip P, McMahon SB, Bradbury EJ
(2006) Chondroitinase ABC promotes sprouting of intact and injured spinal systems
after spinal cord injury. J Neurosci 26:10856-10867.

Bruckner G, Bringmann A, Hartig W, Koppe G, Delpech B, Brauer K (1998) Acute and

long-lasting changes in extracellular-matrix chondroitin-sulphate proteoglycans
induced by injection of chondroitinase ABC in the adult rat brain. Exp Brain Res
121:300-310.

Bruckner G, Hausen D, Hartig W, Drlicek M, Arendt T, Brauer K (1999) Cortical areas

abundant in extracellular matrix chondroitin sulphate proteoglycans are less affected
by cytoskeletal changes in Alzheimer's disease. Neuroscience 92:791-805.

Bruckner G, Brauer K, Hartig W, Wolff JR, Rickmann MJ, Derouiche A, Delpech B, Girard
N, Oertel WH, Reichenbach A (1993) Perineuronal nets provide a polyanionic, glia-
associated form of microenvironment around certain neurons in many parts of the rat
brain. Glia 8:183-200.

Cafferty WB, Yang SH, Duffy PJ, Li S, Strittmatter SM (2007) Functional axonal
regeneration through astrocytic scar genetically modified to digest chondroitin sulfate
proteoglycans. J Neurosci 27:2176-2185.

Chau CH, Shum DK, Li H, Pei J, Lui YY, Wirthlin L, Chan YS, Xu XM (2004)
Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded
guidance channels after spinal cord injury. FASEB J 18:194-196.

Chvatal SA, Kim YT, Bratt-Leal AM, Lee H, Bellamkonda RV (2008) Spatial distribution
and acute anti-inflammatory effects of Methylprednisolone after sustained local
delivery to the contused spinal cord. Biomaterials 29:1967-1975.

Filbin MT (2003) Myelin-associated inhibitors of axonal regeneration in the adult

mammalian CNS. Nat Rev Neurosci 4:703-713.

Galtrey CM, Asher RA, Nothias F, Fawcett JW (2007) Promoting plasticity in the spinal cord
with chondroitinase improves functional recovery after peripheral nerve repair. Brain
130:926-939.

Hamers FP, Lankhorst AJ, van Laar TJ, Veldhuis WB, Gispen WH (2001) Automated
quantitative gait analysis during overground locomotion in the rat: its application to
spinal cord contusion and transection injuries. J Neurotrauma 18:187-201.

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Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988) A new and sensitive method for
measuring thermal nociception in cutaneous hyperalesia. Pain 32:77-88.

Houle JD, Tom VJ, Mayes D, Wagoner G, Phillips N, Silver J (2006) Combining an
autologous peripheral nervous system "bridge" and matrix modification by
chondroitinase allows robust, functional regeneration beyond a hemisection lesion of
the adult rat spinal cord. J Neurosci 26:7405-7415.

Massey JM, Hubscher CH, Wagoner MR, Decker JA, Amps J, Silver J, Onifer SM (2006)
Chondroitinase ABC digestion of the perineuronal net promotes functional collateral
sprouting in the cuneate nucleus after cervical spinal cord injury. J Neurosci 26:4406-
4414.

Massey JM, Amps J, Viapiano MS, Matthews RT, Wagoner MR, Whitaker CM, Alilain W,
Yonkof AL, Khalyfa A, Cooper NG, Silver J, Onifer SM (2008) Increased
chondroitin sulfate proteoglycan expression in denervated brainstem targets following
spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase
ABC and neurotrophin-3. Exp Neurol 209:426-445.

McKeon RJ, Hoke A, Silver J (1995) Injury-induced proteoglycans inhibit the potential for
laminin-mediated axon growth on astrocytic scars. Exp Neurol 136:32-43.

Neumann S, Woolf CJ (1999) Regeneration of dorsal column fibers into and beyond the
lesion site following adult spinal cord injury. Neuron 23:83-91.

Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L (2002) Reactivation of

ocular dominance plasticity in the adult visual cortex. Science 298:1248-1251.

Pizzorusso T, Medini P, Landi S, Baldini S, Berardi N, Maffei L (2006) Structural and

functional recovery from early monocular deprivation in adult rats. Proc Natl Acad
Sci U S A 103:8517-8522.

Schwab JM, Tuli SK, Failli V (2006) The Nogo receptor complex: confining molecules to
molecular mechanisms. Trends Mol Med 12:293-297.

Schwab ME (2004) Nogo and axon regeneration. Curr Opin Neurobiol 14:118-124.

Schwab ME, Bartholdi D (1996) Degeneration and regeneration of axons in the lesioned
spinal cord. Physiol Rev 76:319-370.

Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146-156.

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Tropea D, Caleo M, Maffei L (2003) Synergistic effects of brain-derived neurotrophic factor
and chondroitinase ABC on retinal fiber sprouting after denervation of the superior
colliculus in adult rats. J Neurosci 23:7034-7044.

Vitellaro-Zuccarello L, De Biasi S, Spreafico R (1998) One hundred years of Golgi's

"perineuronal net": history of a denied structure. Ital J Neurol Sci 19:249-253.

Weaver LC, Marsh DR, Gris D, Brown A, Dekaban GA (2006) Autonomic dysreflexia after
spinal cord injury: central mechanisms and strategies for prevention. Prog Brain Res
152:245-263.

Widenfalk J, Lundstromer K, Jubran M, Brene S, Olson L (2001) Neurotrophic factors and

receptors in the immature and adult spinal cord after mechanical injury or kainic acid.
J Neurosci 21:3457-3475.

Woolf CJ, Salter MW (2000) Neuronal plasticity: increasing the gain in pain. Science
288:1765-1769.

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 CHAPTER 6

CONCLUSION AND FUTURE PERSPECTIVES

Our delivery method of chABC provides an alternative treatment to spinal cord injury.

The results showed enhanced axonal sprouting around the lesion site and improved

functional recovery in locomotion. However the ultimate goal is to develop treatment

strategies to promote axonal regrowth through the lesion site to regain functional pathways,

and apply this method for clinical treatment. In this study no axon enters the lesion site and

complete functional recovery was not achieved yet. Therefore, further studies will be

discussed in this chapter to overcome these challenges.

6.1 OPTIMIZATION OF CHABC TREATMENT FOR CLINICAL APPLICATION

To investigate clinical relevance of our method, we need to optimize chABC

treatment. There are several factors that need to be investigated and determined in order to

design the optimized chABC administration: 1) characterize release profile of chABC with

various concentrations of chABC, 2) different lengths of lipid microtube, and 3) then

determine total amount of chABC for a certain delivery period.

For example, with our delivery method the chABC activity was limited to 1 mm

away from lesion site and it is not clear whether the area is a large enough to induce axonal

sprouting and functional improvement in clinical study. Because chABC is a relatively large

molecule, diffusion through neural tissue and extracellular space is limited. chABC delivered

via the hydrogel-microtube system has advantages and diffuses deeper into the tissue (up to

131
1mm) compared to intrathecal delivery of chABC. However in humans, there might be a

need for greater diffusion distances as the CSPG deposition zone may be deeper and larger,

but whether or not chABC diffusion into injured human cord will be a limitation remains to

be determined. The minimum clinically relevant chABC concentration required to elicit

behavioral recovery also remains to be determined.

6.2 EFFECTS OF CHABC ON NERVE TISSUE AND IMMUNE SYSTEM

In this study, single injection of chABC and sustained delivery of chABC did not

increase inflammatory response compared to other controls (no treatment and P‟ase delivery)

after injury. However the effects of chABC on nerve tissue and immune system need to be

considered. When chABC was delivered intrathecally to intact spinal cord, robust sprouting

of descending projects and primary afferents were observed (Barritt et al., 2006). However,

in that study, it did not lead an increase in mechanical allodynia (noxious response to a

usually non-painful stimulus) or thermal hyperalgesia (increased sensitivity to thermal pain).

Other studies also reported that intrathecal delivery of chABC (0.2 ~ 1 ml of 200 U/ml)

showed any morphologic changes in the spinal cord and any neurophysiologic changes in

tibial nerves in rabbits (Olmarker et al., 1991). No adverse effects on nerve tissue and blood

vessels were observed after intrathecal delivery in pig except a slight intrathecal fibrotic

reaction (Olmarker et al., 1996). In those studies, they expected that approximately 1/40

diluted concentration of chABC might be used clinically for chemonucleoysis: however, as

we mentioned previously, the clinically relevant dosage still needs to be determined.

chABC is an enzyme produced from bacteria, so there is a chance to evoke immune

response. In this study, chABC was delivery locally at the lesion site with lipid microtube

132
and hydrogel, however antigenicity and immunogenicity of chABC and the delivery system

still need to be tested. The safety test of chABC for side effects on nerve tissues and immune

response needs to be investigated with the relevant dosage range for clinical application and

sustained delivery system for prolonged delivery period.

6.3 LONGER MICROTUBE: OPTIMIZATION OF DELIVERY VEHICLE

A prolonged sustained delivery would be clinically desirable as increasing the

duration of release agents. To prolong the period of sustained delivery, longer lipid

microtubes can be considered as a delivery vehicle in vivo. The length and properties of

microtube can be controlled by modifying a cooling procedure, varying concentration of lipid

solution and changing ratio between ethanol and water for the solution to dissolve lipid

(Lando et al., 1990; Thomas et al., 1995; Meilander et al., 2001). With the original protocol,

average 37 micron length of microtube is fabricated. The adjustments were made to the

original protocol. 70% ethanol and 1 mg lipid per 1 ml ethanol solution were fixed and a part

of cooling process modified; the rate of decreasing temperature (53-23 deg) changed from

1deg/40min to 1 deg/10 min. The measured average length of microtube was approximately

100 micron (Fig. 6.1). We demonstrated that the length of microtube is controllable and since

the release profile of loaded agents is dependent on the length of microtube, it facilitates to

control drug delivery. The release duration and rate of agent from the delivery scaffold also

can be controlled by modifying the gel. The release profiles are dependent on the type and

concentration of gel (Meilander et al., 2001; Meilander et al., 2003).

133
 50

40
Number of microtubes

30

20

10

0
~40 40-60 60-80 80-100 100-120 120-140 140-160 160-180 180-200 200~

Length of microtubes (µm)

Figure 6.1. Histogram of microtube length distribution with a modified

fabrication procedure. The average length is about 100 µm.

6.4 OPTIMIZATION OF DOSAGE

In this in vivo study presented in Chapter 4 and 5, one concentration/total amount of

chABC and NT-3 was delivered to examine the effect on levels of CSPG deposition, axonal

regeneration and functional recovery after SCI in rats. We demonstrated that there are

significant differences in CSPG digestion, axonal sprouting and functional recovery between

single injection and sustained delivery. Some other studies reported that there is no

significant difference in sprouting axons between chABC treatment and untreated groups

after SCI (Iaci et al., 2007). This implicates that the amount/concentration of delivered

chABC and delivery frequency/period are important to elicit relevant axonal regeneration

134
and behavioral recovery. Therefore, it is necessary to conduct experiments to optimize the

concentration and total amount, and delivery period. The optimal dosage delivered in

combination of chABC and NT-3 also needs to be determined, because combining each

optimal dosage for individual delivery of chABC or NT-3 might not be the best possible

dosage for the combination therapy. For example, other study showed that dramatically

improved (~40 times) axonal sprouting was observed with combination therapy of chABC

and NT-3 (Massey et al., 2008). In this long term in vivo study, even more enhanced axonal

sprouting and functional recovery were observed in chABC/NT-3 treated animals than

individually treated animals. If the concentration and ratio between chABC and NT-3 are

modified, it could synergistically enhance effects on axonal regeneration.

6.5 LONGER IN VIVO STUDY

To evaluate the effects of chABC two animal studies were conducted; a 2 week

study to evaluate of functionality of our chABC-delivery system in Chapter 4 and a 6 week

study to examine axonal sprouting/regeneration and functional recovery as discussed in

Chapter 5. The duration of the in vivo study is a critical factor to determine whether the

administrated therapy is effective or not to treat injuries or diseases. For example, in this

study if we had terminated earlier than 6 weeks, we would not observe the significantly

improved functional recovery in locomotion assessed by stride length between animal groups.

Similarly, if we conduct longer period animal study, such as 8 or 12 weeks, more fibers

might outgrow closer to the lesion and even completely grow through/around the lesion site

to reconstruct synaptic connections. This would lead to improvement of more precise

locomotion controls.

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6.6 RATE AND AMOUNT OF CSPG DEPOSITION IN VIVO AFTER SCI

In the in vivo study, significant differences were observed in the CS-56-IR and 3B3-

IR intensity at 2 weeks, and CS-56-IR intensity at 6 weeks between the chABC-hydrogel-

microtube treated groups and controls. These results suggested that the enzyme, chABC, still

retains its enzymatic activity after being released from the hydrogel-microtube delivery

scaffold in vivo, and digests CSPGs more effectively than the controls, chABC-hydrogel or

chABC-single injection treated groups. However, it is not clear how constant the enzymatic

activity of released chABC remains over this time period and how much chABC is released

from the delivery scaffold and diffused into the tissue. Once chABC is released from the

hydrogel-microtube delivery scaffold, it can be assumed that the activity of chABC will be

similar to fresh, unstabilized chABC, because the ratio of trehalose to chABC becomes a lot

diluted. It is shown that the production of several CSPGs is differentially regulated following

spinal cord injury (Jones et al., 2003) by immunohistological analysis. However, the rates

and amount of CSPG deposition are not known and depend on injury models, degree of

injury and age (Gilbert et al., 2005). Therefore, it would be clinically desirable if the CSPG

deposition rate can be determined for specific injury models and degree of injury.

6.7 COMBINATION STRATEGIES: CHABC AND STEM CELL TRANSPLANTATION

This study showed that combination therapy with thermostabilized chABC and NT-3

significantly enhanced axonal sprouting and functional recovery. Our hydrogel-microtube

delivery system with thermostabilized chABC could provide permissive environment for

neurite outgrowth and can be combined with other strategies, such as delivery of

neurotrophic factor, cell transplantation (NSC, olfactory, Schwann cell, autologus peripheral

136
nerve graft, etc), and acellular scaffold implantation. This combination therapy can be a

potent treatment for many neurodegenerative diseases by promoting axonal regeneration and

restoring complete functional recovery after neuronal injuries. One cell transplantation

strategy, neural stem cell (NSC) therapy, is very promising; however, this approach needs to

be further elucidated before proceeding with clinical trials.

Factors regulating NSC have been investigated, and studies showed that the

modulation of local environment is important in regulating NSC. Evidences have been

provided that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate

decision making between the neuronal and glial lineage. In vitro experiments showed that

CSPGs inhibit migration of neural stem/progenitor cell (NSPC)-derived cells and chABC

treatment attenuated the inhibitory effect (Ikegami et al., 2005; chABC). Also, cell-surface

soluble GAGs involve in internalization of extracellular proteins for some progenitor cell to

facilitate differentiation; an example would be of heparin sulfate proteoglycan for pancreatic

stem cells (Ueda et al., 2008; heparin). As explained above, chABC can be used to modify

the local environment in CNS via digesting CS-GAGs and also to selectively degrade GAGs

for investigating biological roles of CSPGs in vitro.

The use of NSCs in conjunction with chABC may prove to be therapeutic in repairing

CNS by modulating cellular matrix and CSPGs, which act as a barrier for stem cell migration

after transplantation. In many cases, however, transplanted stem cells fail to migrate and

integrate into the host tissue and CSPGs are considered as a putative inhibitor of stem cell

migration in vivo. chABC treatment combined with NSPC transplantation into injured spinal

cord promoted migration of the transplanted cell into the host spinal cord and resulted in

increased sprouting of growth-associated protein -43-positive fibers at the lesion site

137
(Ikegami et al., 2005; chABC). When Müller stem cells were implanted with chABC into

degenerating retina , this resulted in dramatic increase of migration of stem cell into all of

the retina cell layers (Singhal et al., 2008; CSPGs; Fawcett) and differentiation into retinal

neurons and glia (Bull et al., 2008; glaucoma stem).

Studies suggest that chABC treatment can facilitate migration and differentiation of

transplanted NSPCs. Therefore, when the sustained local delivery of thermostabilized

chABC by our hydrogel-microtube system is combined with NSPCs transplantation, it will

be a promising strategy for the regeneration of injured or degenerated CNS tissues. Also, our

chABC treatment can be combined with delivery of other neurotrophic factors and cell

transplantations.

6.8 DIFFERENCES BETWEEN HUMAN CASES AND RAT INJURY MODEL

It is hard to simulate the human cases of SCI in animal experiments: first, human SCI

occurs in the closed vertebral system generally under conscious state and a combination force

of flexion, extension, rotation and compression works on the cord when the injury happens

(Sharma et al., 1993; Choi, 1996). Most human SCI results from fracture of vertebral column

or luxation of vertebrae. In contrast, in animal models, injury or compression is always

conducted on the posterior side of the cord with laminectomy (opened vertebra system) under

anaesthetized state and one force, such as contusion, compression or incision, is applied to

produce injury for high reproducibility (Sharma, 2005a). Therefore, human SCI cases are

more complex and only few cases are similar to experimental models in terms of the

magnitude and severity of the injury (Sharma, 2005b; Onifer et al., 2007). Table 3

summarizes different facts between human and rat spinal cord.

138
 Parameters Human Rat
Number of neurons 109 0.36
Length (cm) 43-45 8-10
Weight (g) 34 0.7
Proportion to brain
2 35
(volume %)
Glial/neuron ratio 12-16 10-14
2438 50*
Weight drop injury (g) 971 20*
485 10*

Table 3. A comparison between human and rat spinal cord.

* Weight for rat SCI is about one fifth to human SCI. Table from
Sharma, 2007.

Our study required an SCI model allowing for behavioral and immunohistological

analysis to examine functional recovery and axonal regeneration. There are several

commonly used models for spinal cord injury and each has advantages and disadvantages:

complete spinal transection, dorsal over hemisection, lateral hemisection injury, compression

and contusion.

The complete transection model removes any overlap between spared and regenerated

fibers. However, the animal undergoes severe pain and various syndromes, such as bowel

and bladder dysfunction and no functional recovery with hindlimbs, because the transection

completely eliminates functions below the injury site. Therefore, it requires relatively long

term management with various post-surgical complications and morbidity is higher than

other injury models.

The contusion model could be the most clinically relevant model, although it still

lacks some of the absent factors described above. A fluid filled cyst forms at the impacted

area and is surrounded by tissue containing spared/intact axons (Kwon et al., 2002). Because
139
of the spared axons around the cyst, it is hard to distinguish between spared and regenerated

axons (Steward et al., 2003) and it is a limitation for studies focusing on regeneration.

The lateral transection model is a complete transection of one side of the spinal cord.

In this model all pathways in one side of the cord are severed. However, intact axons in the

non-lesioned side can sprout into the lesioned side and make it hard to distinguish from

regenerated axons (Kwon et al., 2002).

The dorsal over hemisection model has relatively moderate severity of injury. In this

injury model, damage is limited in the dorsal column and animals undergo less stress and

pain with less post-surgery dysfunctions than other injury models. Axonal regeneration can

be easily quantified by injection of anterograde tracer (i.e. BDA) to the corticospinal tract

(CST) or retrograde tracer to the primary sensory pathway (i.e. cholera toxin subunit B), and

spared fiber is not a critical issue. Therefore, the dorsal over hemisection model was chosen

for our study. There is a chance of spared fibers in the bilateral transected CST (Vavrek et al.,

2006), and the primary sensory pathway was chosen in this study to examine regenerating

axons.

As a further study, contusion injury model can be used to examine the delivery

efficiency of our topical delivery method with chABC and develop a more relevant delivery

method for human therapy. In the contusion model, it would be difficult to deliver drugs deep

through the surrounding tissue to the cyst with our topical delivery model if the drug is not

small enough. Diffusion efficiency of chABC needs to be examined in this model. If the

diffusion is not enough to remove CSPGs and promote axonal outgrowth, alternative delivery

methods should be developed, such as a direct injection of mitcrotube-trehalose/chABC into

the cyst by needle.

140
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(2006) Chondroitinase ABC promotes sprouting of intact and injured spinal systems
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Bull ND, Limb GA, Martin KR (2008) Human Muller stem cell (MIO-M1) transplantation in
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